The Ty1-copia LTR retroelement family PARTC is highly conserved in conifers over 200 MY of evolution.

PubMed

Zuccolo, Andrea; Scofield, Douglas G; De Paoli, Emanuele; Morgante, Michele

2015-08-15

Long Terminal Repeat retroelements (LTR-RTs) are a major component of many plant genomes. Although well studied and described in angiosperms, their features and dynamics are poorly understood in gymnosperms. Representative complete copies of a Ty1-copia element isolate in Picea abies and named PARTC were identified in six other conifer species (Picea glauca, Pinus sylvestris, Pinus taeda, Abies sibirica, Taxus baccata and Juniperus communis) covering more than 200 million years of evolution. Here we characterized the structure of this element, assessed its abundance across conifers, studied the modes and timing of its amplification, and evaluated the degree of conservation of its extant copies at nucleotide level over distant species. We demonstrated that the element is ancient, abundant, widespread and its paralogous copies are present in the genera Picea, Pinus and Abies as an LTR-RT family. The amplification leading to the extant copies of PARTC occurred over long evolutionary times spanning 10s of MY and mostly took place after the speciation of the conifers analyzed. The level of conservation of PARTC is striking and may be explained by low substitution rates and limited removal mechanisms for LTR-RTs. These PARTC features and dynamics are representative of a more general scenario for LTR-RTs in gymnosperms quite different from that characterizing the vast majority of LTR-RT elements in angiosperms. Copyright © 2015 Elsevier B.V. All rights reserved.

Recent Amplification of the Kangaroo Endogenous Retrovirus, KERV, Limited to the Centromere▿

PubMed Central

Ferreri, Gianni C.; Brown, Judith D.; Obergfell, Craig; Jue, Nathaniel; Finn, Caitlin E.; O'Neill, Michael J.; O'Neill, Rachel J.

2011-01-01

Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events. PMID:21389136

Phylogenomics of nonavian reptiles and the structure of the ancestral amniote genome

PubMed Central

Shedlock, Andrew M.; Botka, Christopher W.; Zhao, Shaying; Shetty, Jyoti; Zhang, Tingting; Liu, Jun S.; Deschavanne, Patrick J.; Edwards, Scott V.

2007-01-01

We report results of a megabase-scale phylogenomic analysis of the Reptilia, the sister group of mammals. Large-scale end-sequence scanning of genomic clones of a turtle, alligator, and lizard reveals diverse, mammal-like landscapes of retroelements and simple sequence repeats (SSRs) not found in the chicken. Several global genomic traits, including distinctive phylogenetic lineages of CR1-like long interspersed elements (LINEs) and a paucity of A-T rich SSRs, characterize turtles and archosaur genomes, whereas higher frequencies of tandem repeats and a lower global GC content reveal mammal-like features in Anolis. Nonavian reptile genomes also possess a high frequency of diverse and novel 50-bp unit tandem duplications not found in chicken or mammals. The frequency distributions of ≈65,000 8-mer oligonucleotides suggest that rates of DNA-word frequency change are an order of magnitude slower in reptiles than in mammals. These results suggest a diverse array of interspersed and SSRs in the common ancestor of amniotes and a genomic conservatism and gradual loss of retroelements in reptiles that culminated in the minimalist chicken genome. PMID:17307883

Plant centromeres.

PubMed

Lamb, J C; Yu, W; Han, F; Birchler, J A

2008-01-01

Plant centromeres are generally composed of tandem arrays of simple repeats that are typical of a particular species, but that evolve rapidly. Centromere specific retroelements are also present. These arrays associate with a centromere specific variant of histone H3 that anchors the site of the kinetochore. Although such DNA arrays are typical of the centromere, the specification of centromere activity has an epigenetic component as shown by the fact that centromeres are formed in the absence of such repeats and that centromeres in dicentric chromosomes regularly undergo inactivation.

Molecular structure and chromosome distribution of three repetitive DNA families in Anemone hortensis L. (Ranunculaceae).

PubMed

Mlinarec, Jelena; Chester, Mike; Siljak-Yakovlev, Sonja; Papes, Drazena; Leitch, Andrew R; Besendorfer, Visnja

2009-01-01

The structure, abundance and location of repetitive DNA sequences on chromosomes can characterize the nature of higher plant genomes. Here we report on three new repeat DNA families isolated from Anemone hortensis L.; (i) AhTR1, a family of satellite DNA (stDNA) composed of a 554-561 bp long EcoRV monomer; (ii) AhTR2, a stDNA family composed of a 743 bp long HindIII monomer and; (iii) AhDR, a repeat family composed of a 945 bp long HindIII fragment that exhibits some sequence similarity to Ty3/gypsy-like retroelements. Fluorescence in-situ hybridization (FISH) to metaphase chromosomes of A. hortensis (2n = 16) revealed that both AhTR1 and AhTR2 sequences co-localized with DAPI-positive AT-rich heterochromatic regions. AhTR1 sequences occur at intercalary DAPI bands while AhTR2 sequences occur at 8-10 terminally located heterochromatic blocks. In contrast AhDR sequences are dispersed over all chromosomes as expected of a Ty3/gypsy-like element. AhTR2 and AhTR1 repeat families include polyA- and polyT-tracks, AT/TA-motifs and a pentanucleotide sequence (CAAAA) that may have consequences for chromatin packing and sequence homogeneity. AhTR2 repeats also contain TTTAGGG motifs and degenerate variants. We suggest that they arose by interspersion of telomeric repeats with subtelomeric repeats, before hybrid unit(s) amplified through the heterochromatic domain. The three repetitive DNA families together occupy approximately 10% of the A. hortensis genome. Comparative analyses of eight Anemone species revealed that the divergence of the A. hortensis genome was accompanied by considerable modification and/or amplification of repeats.

Repression of chimeric transcripts emanating from endogenous retrotransposons by a sequence-specific transcription factor

PubMed Central

2014-01-01

Background Retroviral elements are pervasively transcribed and dynamically regulated during development. While multiple histone- and DNA-modifying enzymes have broadly been associated with their global silencing, little is known about how the many diverse retroviral families are each selectively recognized. Results Here we show that the zinc finger protein Krüppel-like Factor 3 (KLF3) specifically silences transcription from the ORR1A0 long terminal repeat in murine fetal and adult erythroid cells. In the absence of KLF3, we detect widespread transcription from ORR1A0 elements driven by the master erythroid regulator KLF1. In several instances these aberrant transcripts are spliced to downstream genic exons. One such chimeric transcript produces a novel, dominant negative isoform of PU.1 that can induce erythroid differentiation. Conclusions We propose that KLF3 ensures the integrity of the murine erythroid transcriptome through the selective repression of a particular retroelement and is likely one of multiple sequence-specific factors that cooperate to achieve global silencing. PMID:24946810

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

2011-01-01

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis▿

PubMed Central

Hizi, Amnon

2008-01-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5′ end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3′ end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs. PMID:18753200

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

The reverse transcriptase of the Tf1 retrotransposon has a specific novel activity for generating the RNA self-primer that is functional in cDNA synthesis.

PubMed

Hizi, Amnon

2008-11-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5' end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3' end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

PubMed

Arkhipova, Irina R; Yushenova, Irina A; Rodriguez, Fernando

2017-09-01

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Placental Hypomethylation Is More Pronounced in Genomic Loci Devoid of Retroelements

PubMed Central

Chatterjee, Aniruddha; Macaulay, Erin C.; Rodger, Euan J.; Stockwell, Peter A.; Parry, Matthew F.; Roberts, Hester E.; Slatter, Tania L.; Hung, Noelyn A.; Devenish, Celia J.; Morison, Ian M.

2016-01-01

The human placenta is hypomethylated compared to somatic tissues. However, the degree and specificity of placental hypomethylation across the genome is unclear. We assessed genome-wide methylation of the human placenta and compared it to that of the neutrophil, a representative homogeneous somatic cell. We observed global hypomethylation in placenta (relative reduction of 22%) compared to neutrophils. Placental hypomethylation was pronounced in intergenic regions and gene bodies, while the unmethylated state of the promoter remained conserved in both tissues. For every class of repeat elements, the placenta showed lower methylation but the degree of hypomethylation differed substantially between these classes. However, some retroelements, especially the evolutionarily younger Alu elements, retained high levels of placental methylation. Surprisingly, nonretrotransposon-containing sequences showed a greater degree of placental hypomethylation than retrotransposons in every genomic element (intergenic, introns, and exons) except promoters. The differentially methylated fragments (DMFs) in placenta and neutrophils were enriched in gene-poor and CpG-poor regions. The placentally hypomethylated DMFs were enriched in genomic regions that are usually inactive, whereas hypermethylated DMFs were enriched in active regions. Hypomethylation of the human placenta is not specific to retroelements, indicating that the evolutionary advantages of placental hypomethylation go beyond those provided by expression of retrotransposons and retrogenes. PMID:27172225

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

PubMed

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-07-01

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Woot, an Active Gypsy-Class Retrotransposon in the Flour Beetle, Tribolium Castaneum, Is Associated with a Recent Mutation

PubMed Central

Beeman, R. W.; Thomson, M. S.; Clark, J. M.; DeCamillis, M. A.; Brown, S. J.; Denell, R. E.

1996-01-01

A recently isolated, lethal mutation of the homeotic Abdominal gene of the red flour beetle Tribolium castaneum is associated with an insertion of a novel retrotransposon into an intron. Sequence analysis indicates that this retrotransposon, named Woot, is a member of the gypsy family of mobile elements. Most strains of T. castaneum appear to harbor ~25-35 copies of Woot per genome. Woot is composed of long terminal repeats of unprecedented length (3.6 kb each), flanking an internal coding region 5.0 kb in length. For most copies of Woot, the internal region includes two open reading frames (ORFs) that correspond to the gag and pol genes of previously described retrotransposons and retroviruses. The copy of Woot inserted into Abdominal bears an apparent single frameshift mutation that separates the normal second ORF into two. Woot does not appear to generate infectious virions by the criterion that no envelop gene is discernible. The association of Woot with a recent mutation suggests that this retroelement is currently transpositionally active in at least some strains. PMID:8722793

The Gypsy Database (GyDB) of mobile genetic elements

PubMed Central

Lloréns, C.; Futami, R.; Bezemer, D.; Moya, A.

2008-01-01

In this article, we introduce the Gypsy Database (GyDB) of mobile genetic elements, an in-progress database devoted to the non-redundant analysis and evolutionary-based classification of mobile genetic elements. In this first version, we contemplate eukaryotic Ty3/Gypsy and Retroviridae long terminal repeats (LTR) retroelements. Phylogenetic analyses based on the gag-pro-pol internal region commonly presented by these two groups strongly support a certain number of previously described Ty3/Gypsy lineages originally reported from reverse-transcriptase (RT) analyses. Vertebrate retroviruses (Retroviridae) are also constituted in several monophyletic groups consistent with genera proposed by the ICTV nomenclature, as well as with the current tendency to classify both endogenous and exogenous retroviruses by three major classes (I, II and III). Our inference indicates that all protein domains codified by the gag-pro-pol internal region of these two groups agree in a collective presentation of a particular evolutionary history, which may be used as a main criterion to differentiate their molecular diversity in a comprehensive collection of phylogenies and non-redundant molecular profiles useful in the identification of new Ty3/Gypsy and Retroviridae species. The GyDB project is available at http://gydb.uv.es. PMID:17895280

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

PubMed

Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

2016-01-01

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

PubMed Central

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-01-01

Background and Aims The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. Methods A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Key Results Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. Conclusions The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species. PMID:23666888

Involvement of Ethylene in Stress-Induced Expression of the TLC1.1 Retrotransposon from Lycopersicon chilense Dun.1[w

PubMed Central

Tapia, Gerardo; Verdugo, Isabel; Yañez, Mónica; Ahumada, Iván; Theoduloz, Cristina; Cordero, Cecilia; Poblete, Fernando; González, Enrique; Ruiz-Lara, Simón

2005-01-01

The TLC1 family is one of the four families of long terminal repeat (LTR) retrotransposons identified in the genome of Lycopersicon chilense. Here, we show that this family of retroelements is transcriptionally active and its expression is induced in response to diverse stress conditions such as wounding, protoplast preparation, and high salt concentrations. Several stress-associated signaling molecules, including ethylene, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid, are capable of inducing TLC1 family expression in vivo. A representative of this family, named TLC1.1, was isolated from a genomic library from L. chilense. Transient expression assays in leaf protoplasts and stably transformed tobacco (Nicotiana tabacum) plants demonstrate that the U3 domain of the 5′-LTR region of this element can drive stress-induced transcriptional activation of the β-glucuronidase reporter gene. Two 57-bp tandem repeated sequences are found in this region, including an 8-bp motif, ATTTCAAA, previously identified as an ethylene-responsive element box in the promoter region of ethylene-induced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the β-glucuronidase gene shows that these elements are required for ethylene-responsive gene expression in protoplasts and transgenic plants. We suggest that ethylene-dependent signaling is the main signaling pathway involved in the regulation of the expression of the TLC1.1 element from L. chilense. PMID:16040666

A comparative study of retrotransposons in the centromeric regions of A and B chromosomes of maize.

PubMed

Theuri, J; Phelps-Durr, T; Mathews, S; Birchler, J

2005-01-01

Bacterial Artificial Chromosomes (BACs) derived from the B chromosome, based on homology with the B specific sequence, were subcloned and sequenced. Analysis of DNA sequence data indicated the presence of 23 common retroelements, as well as novel sequences of B chromosome origin. Generally, where the same retrotransposon type was observed in both A and B chromosomes, there were more copies per unit of sequence in the B centromeric region (the major site of B repeat) than in the A centromere, except for Huck-1. Based on previous estimates of the age of the major burst of transposition into the maize genome, the oldest retrotransposons (Ji-6 and Tekay, approximately 5.0 and 5.2 million years ago, respectively) were found in the B centromere region only, while the next two oldest (Huck-1 and Opie-1) were found in both the A and B sequences. Phylogenetic analysis of Opie retroelements from both A and B centromeres indicated that some of the B Opie centromeric sequences share a more recent common ancestor with A Opie retroelements than they do with other B Opie centromeric sequences. These results imply that the supernumerary maize B chromosome has coexisted with the A chromosomes during that period of transposition. They also support the hypothesis that the B chromosome had its origins from A chromosome elements, or that alternative origins, such as being donated to the maize genome in a wide species cross, preceded six million years ago, because the spectrum of retrotransposons in the two chromosomes is quite similar.

Retroelements and their impact on genome evolution and functioning.

PubMed

Gogvadze, Elena; Buzdin, Anton

2009-12-01

Retroelements comprise a considerable fraction of eukaryotic genomes. Since their initial discovery by Barbara McClintock in maize DNA, retroelements have been found in genomes of almost all organisms. First considered as a "junk DNA" or genomic parasites, they were shown to influence genome functioning and to promote genetic innovations. For this reason, they were suggested as an important creative force in the genome evolution and adaptation of an organism to altered environmental conditions. In this review, we summarize the up-to-date knowledge of different ways of retroelement involvement in structural and functional evolution of genes and genomes, as well as the mechanisms generated by cells to control their retrotransposition.

DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

PubMed

Knisbacher, Binyamin A; Levanon, Erez Y

2015-04-01

Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution. © 2015 New York Academy of Sciences.

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean.

PubMed

Innes, Roger W; Ameline-Torregrosa, Carine; Ashfield, Tom; Cannon, Ethalinda; Cannon, Steven B; Chacko, Ben; Chen, Nicolas W G; Couloux, Arnaud; Dalwani, Anita; Denny, Roxanne; Deshpande, Shweta; Egan, Ashley N; Glover, Natasha; Hans, Christian S; Howell, Stacy; Ilut, Dan; Jackson, Scott; Lai, Hongshing; Mammadov, Jafar; Del Campo, Sara Martin; Metcalf, Michelle; Nguyen, Ashley; O'Bleness, Majesta; Pfeil, Bernard E; Podicheti, Ram; Ratnaparkhe, Milind B; Samain, Sylvie; Sanders, Iryna; Ségurens, Béatrice; Sévignac, Mireille; Sherman-Broyles, Sue; Thareau, Vincent; Tucker, Dominic M; Walling, Jason; Wawrzynski, Adam; Yi, Jing; Doyle, Jeff J; Geffroy, Valérie; Roe, Bruce A; Maroof, M A Saghai; Young, Nevin D

2008-12-01

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.

CR1 clade of non-LTR retrotransposons from Maculinea butterflies (Lepidoptera: Lycaenidae): evidence for recent horizontal transmission

PubMed Central

Novikova, Olga; Śliwińska, Ewa; Fet, Victor; Settele, Josef; Blinov, Alexander; Woyciechowski, Michal

2007-01-01

Background Non-long terminal repeat (non-LTR) retrotransposons are mobile genetic elements that propagate themselves by reverse transcription of an RNA intermediate. Non-LTR retrotransposons are known to evolve mainly via vertical transmission and random loss. Horizontal transmission is believed to be a very rare event in non-LTR retrotransposons. Our knowledge of distribution and diversity of insect non-LTR retrotransposons is limited to a few species – mainly model organisms such as dipteran genera Drosophila, Anopheles, and Aedes. However, diversity of non-LTR retroelements in arthropods seems to be much richer. The present study extends the analysis of non-LTR retroelements to CR1 clade from four butterfly species of genus Maculinea (Lepidoptera: Lycaenidae). The lycaenid genus Maculinea, the object of interest for evolutionary biologists and also a model group for European biodiversity studies, possesses a unique, specialized myrmecophilous lifestyle at larval stage. Their caterpillars, after three weeks of phytophagous life on specific food plants drop to the ground where they are adopted to the ant nest by Myrmica foraging workers. Results We found that the genome of Maculinea butterflies contains multiple CR1 lineages of non-LTR retrotransposons, including those from MacCR1A, MacCR1B and T1Q families. A comparative analysis of RT nucleotide sequences demonstrated an extremely high similarity among elements both in interspecific and intraspecific comparisons. CR1A-like elements were found only in family Lycaenidae. In contrast, MacCR1B lineage clones were extremely similar to CR1B non-LTR retrotransposons from Bombycidae moths: silkworm Bombyx mori and Oberthueria caeca. Conclusion The degree of coding sequence similarity of the studied elements, their discontinuous distribution, and results of divergence-versus-age analysis make it highly unlikely that these sequences diverged at the same time as their host taxa. The only reasonable alternative explanation is horizontal transfer. In addition, phylogenetic markers for population analysis of Maculinea could be developed based on the described non-LTR retrotransposons. PMID:17588269

Drosophila: Retrotransposons Making up Telomeres.

PubMed

Casacuberta, Elena

2017-07-19

Drosophila and extant species are the best-studied telomerase exception. In this organism, telomere elongation is coupled with targeted retrotransposition of Healing Transposon (HeT-A) and Telomere Associated Retrotransposon (TART) with sporadic additions of Telomere Associated and HeT-A Related (TAHRE), all three specialized non-Long Terminal Repeat (non-LTR) retrotransposons. These three very special retroelements transpose in head to tail arrays, always in the same orientation at the end of the chromosomes but never in interior locations. Apparently, retrotransposon and telomerase telomeres might seem very different, but a detailed view of their mechanisms reveals similarities explaining how the loss of telomerase in a Drosophila ancestor could successfully have been replaced by the telomere retrotransposons. In this review, we will discover that although HeT-A, TART, and TAHRE are still the only examples to date where their targeted transposition is perfectly tamed into the telomere biology of Drosophila, there are other examples of retrotransposons that manage to successfully integrate inside and at the end of telomeres. Because the aim of this special issue is viral integration at telomeres, understanding the base of the telomerase exceptions will help to obtain clues on similar strategies that mobile elements and viruses could have acquired in order to ensure their survival in the host genome.

Retroelements (LINEs and SINEs) in vole genomes: differential distribution in the constitutive heterochromatin.

PubMed

Acosta, M J; Marchal, J A; Fernández-Espartero, C H; Bullejos, M; Sánchez, A

2008-01-01

The chromosomal distribution of mobile genetic elements is scarcely known in Arvicolinae species, but could be of relevance to understand the origin and complex evolution of the sex chromosome heterochromatin. In this work we cloned two retrotransposon sequences, L1 and SINE-B1, from the genome of Chionomys nivalis and investigated their chromosomal distribution on several arvicoline species. Our results demonstrate first that both retroelements are the most abundant repeated DNA sequences in the genome of these species. L1 elements, in most species, are highly accumulated in the sex chromosomes compared to the autosomes. This favoured L1 insertion could have played an important role in the origin of the enlarged heterochromatic blocks existing in the sex chromosomes of some Microtus species. Also, we propose that L1 accumulation on the X heterochromatin could have been the consequence of different, independent and rapid amplification processes acting in each species. SINE elements, however, were completely lacking from the constitutive heterochromatin, either in autosomes or in the heterochromatic blocks of sex chromosomes. These data could indicate that some SINE elements are incompatible with the formation of heterochromatic complexes and hence are necessarily missing from the constitutive heterochromatin.

Diversity, distribution and dynamics of full-length Copia and Gypsy LTR retroelements in Solanum lycopersicum.

PubMed

Paz, Rosalía Cristina; Kozaczek, Melisa Eliana; Rosli, Hernán Guillermo; Andino, Natalia Pilar; Sanchez-Puerta, Maria Virginia

2017-10-01

Transposable elements are the most abundant components of plant genomes and can dramatically induce genetic changes and impact genome evolution. In the recently sequenced genome of tomato (Solanum lycopersicum), the estimated fraction of elements corresponding to retrotransposons is nearly 62%. Given that tomato is one of the most important vegetable crop cultivated and consumed worldwide, understanding retrotransposon dynamics can provide insight into its evolution and domestication processes. In this study, we performed a genome-wide in silico search of full-length LTR retroelements in the tomato nuclear genome and annotated 736 full-length Gypsy and Copia retroelements. The dispersion level across the 12 chromosomes, the diversity and tissue-specific expression of those elements were estimated. Phylogenetic analysis based on the retrotranscriptase region revealed the presence of 12 major lineages of LTR retroelements in the tomato genome. We identified 97 families, of which 77 and 20 belong to the superfamilies Copia and Gypsy, respectively. Each retroelement family was characterized according to their element size, relative frequencies and insertion time. These analyses represent a valuable resource for comparative genomics within the Solanaceae, transposon-tagging and for the design of cultivar-specific molecular markers in tomato.

Evolution of Centromeric Retrotransposons in Grasses

PubMed Central

Sharma, Anupma; Presting, Gernot G.

2014-01-01

Centromeric retrotransposons (CRs) constitute a family of plant retroelements, some of which have the ability to target their insertion almost exclusively to the functional centromeres. Our exhaustive analysis of CR family members in four grass genomes revealed not only horizontal transfer (HT) of CR elements between the oryzoid and panicoid grass lineages but also their subsequent recombination with endogenous elements that in some cases created prolific recombinants in foxtail millet and sorghum. HT events are easily identifiable only in cases where host genome divergence significantly predates HT, thus documented HT events likely represent only a fraction of the total. If the more difficult to detect ancient HT events occurred at frequencies similar to those observable in present day grasses, the extant long terminal repeat retrotransposons represent the mosaic products of HT and recombination that are optimized for retrotransposition in their host genomes. This complicates not only phylogenetic analysis but also the establishment of a meaningful retrotransposon nomenclature, which we have nevertheless attempted to implement here. In contrast to the plant-centric naming convention used currently for CR elements, we classify elements primarily based on their phylogenetic relationships regardless of host plant, using the exhaustively studied maize elements assigned to six different subfamilies as a standard. The CR2 subfamily is the most widely distributed of the six CR subfamilies discovered in grass genomes to date and thus the most likely to play a functional role at grass centromeres. PMID:24814286

Tyrosine Recombinase Retrotransposons and Transposons.

PubMed

Poulter, Russell T M; Butler, Margi I

2015-04-01

Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes.

Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

PubMed

Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

2017-03-01

To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human spermatozoa; and 4) de novo retrotransposition events occur in human spermatozoa. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Positive Selection and Multiple Losses of the LINE-1-Derived L1TD1 Gene in Mammals Suggest a Dual Role in Genome Defense and Pluripotency

PubMed Central

Yang, Lei; Neme, Rafik; Wichman, Holly A.; Malik, Harmit S.

2014-01-01

Mammalian genomes comprise many active and fossilized retroelements. The obligate requirement for retroelement integration affords host genomes an opportunity to ‘domesticate’ retroelement genes for their own purpose, leading to important innovations in genome defense and placentation. While many such exaptations involve retroviruses, the L1TD1 gene is the only known domesticated gene whose protein-coding sequence is almost entirely derived from a LINE-1 (L1) retroelement. Human L1TD1 has been shown to play an important role in pluripotency maintenance. To investigate how this role was acquired, we traced the origin and evolution of L1TD1. We find that L1TD1 originated in the common ancestor of eutherian mammals, but was lost or pseudogenized multiple times during mammalian evolution. We also find that L1TD1 has evolved under positive selection during primate and mouse evolution, and that one prosimian L1TD1 has ‘replenished’ itself with a more recent L1 ORF1 from the prosimian genome. These data suggest that L1TD1 has been recurrently selected for functional novelty, perhaps for a role in genome defense. L1TD1 loss is associated with L1 extinction in several megabat lineages, but not in sigmodontine rodents. We hypothesize that L1TD1 could have originally evolved for genome defense against L1 elements. Later, L1TD1 may have become incorporated into pluripotency maintenance in some lineages. Our study highlights the role of retroelement gene domestication in fundamental aspects of mammalian biology, and that such domesticated genes can adopt different functions in different lineages. PMID:25211013

Hydroquinone induces DNA hypomethylation-independent overexpression of retroelements in human leukemia and hematopoietic stem cells.

PubMed

Conti, Anastasia; Rota, Federica; Ragni, Enrico; Favero, Chiara; Motta, Valeria; Lazzari, Lorenza; Bollati, Valentina; Fustinoni, Silvia; Dieci, Giorgio

2016-06-10

Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzene carcinogenesis studies. Copyright © 2016 Elsevier Inc. All rights reserved.

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA

PubMed Central

Naorem, Santa S.; Han, Jin; Wang, Shufang; Lee, William R.; Heng, Xiao; Miller, Jeff F.

2017-01-01

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation. PMID:29109248

Short interspersed transposable elements (SINEs) are excluded from imprinted regions in the human genome.

PubMed

Greally, John M

2002-01-08

To test whether regions undergoing genomic imprinting have unique genomic characteristics, imprinted and nonimprinted human loci were compared for nucleotide and retroelement composition. Maternally and paternally expressed subgroups of imprinted genes were found to differ in terms of guanine and cytosine, CpG, and retroelement content, indicating a segregation into distinct genomic compartments. Imprinted regions have been normally permissive to L1 long interspersed transposable element retroposition during mammalian evolution but universally and significantly lack short interspersed transposable elements (SINEs). The primate-specific Alu SINEs, as well as the more ancient mammalian-wide interspersed repeat SINEs, are found at significantly low densities in imprinted regions. The latter paleogenomic signature indicates that the sequence characteristics of currently imprinted regions existed before the mammalian radiation. Transitions from imprinted to nonimprinted genomic regions in cis are characterized by a sharp inflection in SINE content, demonstrating that this genomic characteristic can help predict the presence and extent of regions undergoing imprinting. During primate evolution, SINE accumulation in imprinted regions occurred at a decreased rate compared with control loci. The constraint on SINE accumulation in imprinted regions may be mediated by an active selection process. This selection could be because of SINEs attracting and spreading methylation, as has been found at other loci. Methylation-induced silencing could lead to deleterious consequences at imprinted loci, where inactivation of one allele is already established, and expression is often essential for embryonic growth and survival.

Short interspersed transposable elements (SINEs) are excluded from imprinted regions in the human genome

PubMed Central

Greally, John M.

2002-01-01

To test whether regions undergoing genomic imprinting have unique genomic characteristics, imprinted and nonimprinted human loci were compared for nucleotide and retroelement composition. Maternally and paternally expressed subgroups of imprinted genes were found to differ in terms of guanine and cytosine, CpG, and retroelement content, indicating a segregation into distinct genomic compartments. Imprinted regions have been normally permissive to L1 long interspersed transposable element retroposition during mammalian evolution but universally and significantly lack short interspersed transposable elements (SINEs). The primate-specific Alu SINEs, as well as the more ancient mammalian-wide interspersed repeat SINEs, are found at significantly low densities in imprinted regions. The latter paleogenomic signature indicates that the sequence characteristics of currently imprinted regions existed before the mammalian radiation. Transitions from imprinted to nonimprinted genomic regions in cis are characterized by a sharp inflection in SINE content, demonstrating that this genomic characteristic can help predict the presence and extent of regions undergoing imprinting. During primate evolution, SINE accumulation in imprinted regions occurred at a decreased rate compared with control loci. The constraint on SINE accumulation in imprinted regions may be mediated by an active selection process. This selection could be because of SINEs attracting and spreading methylation, as has been found at other loci. Methylation-induced silencing could lead to deleterious consequences at imprinted loci, where inactivation of one allele is already established, and expression is often essential for embryonic growth and survival. PMID:11756672

Network dynamics of eukaryotic LTR retroelements beyond phylogenetic trees

PubMed Central

Llorens, Carlos; Muñoz-Pomer, Alfonso; Bernad, Lucia; Botella, Hector; Moya, Andrés

2009-01-01

Background Sequencing projects have allowed diverse retroviruses and LTR retrotransposons from different eukaryotic organisms to be characterized. It is known that retroviruses and other retro-transcribing viruses evolve from LTR retrotransposons and that this whole system clusters into five families: Ty3/Gypsy, Retroviridae, Ty1/Copia, Bel/Pao and Caulimoviridae. Phylogenetic analyses usually show that these split into multiple distinct lineages but what is yet to be understood is how deep evolution occurred in this system. Results We combined phylogenetic and graph analyses to investigate the history of LTR retroelements both as a tree and as a network. We used 268 non-redundant LTR retroelements, many of them introduced for the first time in this work, to elucidate all possible LTR retroelement phylogenetic patterns. These were superimposed over the tree of eukaryotes to investigate the dynamics of the system, at distinct evolutionary times. Next, we investigated phenotypic features such as duplication and variability of amino acid motifs, and several differences in genomic ORF organization. Using this information we characterized eight reticulate evolution markers to construct phenotypic network models. Conclusion The evolutionary history of LTR retroelements can be traced as a time-evolving network that depends on phylogenetic patterns, epigenetic host-factors and phenotypic plasticity. The Ty1/Copia and the Ty3/Gypsy families represent the oldest patterns in this network that we found mimics eukaryotic macroevolution. The emergence of the Bel/Pao, Retroviridae and Caulimoviridae families in this network can be related with distinct inflations of the Ty3/Gypsy family, at distinct evolutionary times. This suggests that Ty3/Gypsy ancestors diversified much more than their Ty1/Copia counterparts, at distinct geological eras. Consistent with the principle of preferential attachment, the connectivities among phenotypic markers, taken as network-represented combinations, are power-law distributed. This evidences an inflationary mode of evolution where the system diversity; 1) expands continuously alternating vertical and gradual processes of phylogenetic divergence with episodes of modular, saltatory and reticulate evolution; 2) is governed by the intrinsic capability of distinct LTR retroelement host-communities to self-organize their phenotypes according to emergent laws characteristic of complex systems. Reviewers This article was reviewed by Eugene V. Koonin, Eric Bapteste, and Enmanuelle Lerat (nominated by King Jordan) PMID:19883502

Relationships of gag-pol diversity between Ty3/Gypsy and Retroviridae LTR retroelements and the three kings hypothesis

PubMed Central

2008-01-01

Background The origin of vertebrate retroviruses (Retroviridae) is yet to be thoroughly investigated, but due to their similarity and identical gag-pol (and env) genome structure, it is accepted that they evolve from Ty3/Gypsy LTR retroelements the retrotransposons and retroviruses of plants, fungi and animals. These 2 groups of LTR retroelements code for 3 proteins rarely studied due to the high variability – gag polyprotein, protease and GPY/F module. In relation to 3 previously proposed Retroviridae classes I, II and II, investigation of the above proteins conclusively uncovers important insights regarding the ancient history of Ty3/Gypsy and Retroviridae LTR retroelements. Results We performed a comprehensive study of 120 non-redundant Ty3/Gypsy and Retroviridae LTR retroelements. Phylogenetic reconstruction inferred based on the concatenated analysis of the gag and pol polyproteins shows a robust phylogenetic signal regarding the clustering of OTUs. Evaluation of gag and pol polyproteins separately yields discordant information. While pol signal supports the traditional perspective (2 monophyletic groups), gag polyprotein describes an alternative scenario where each Retroviridae class can be distantly related with one or more Ty3/Gypsy lineages. We investigated more in depth this evidence through comparative analyses performed based on the gag polyprotein, the protease and the GPY/F module. Our results indicate that contrary to the traditional monophyletic view of the origin of vertebrate retroviruses, the Retroviridae class I is a molecular fossil, preserving features that were probably predominant among Ty3/Gypsy ancestors predating the split of plants, fungi and animals. In contrast, classes II and III maintain other phenotypes that emerged more recently during Ty3/Gypsy evolution. Conclusion The 3 Retroviridae classes I, II and III exhibit phenotypic differences that delineate a network never before reported between Ty3/Gypsy and Retroviridae LTR retroelements. This new scenario reveals how the diversity of vertebrate retroviruses is polyphyletically recurrent into the Ty3/Gypsy evolution, i.e. older than previously thought. The simplest hypothesis to explain this finding is that classes I, II and III trace back to at least 3 Ty3/Gypsy ancestors that emerged at different evolutionary times prior to protostomes-deuterostomes divergence. We have called this "the three kings hypothesis" concerning the origin of vertebrate retroviruses. PMID:18842133

LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase

PubMed Central

Metzner, Mirjam; Jäck, Hans-Martin; Wabl, Matthias

2012-01-01

LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus. PMID:23133680

Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy.

PubMed

Gabus, Caroline; Ivanyi-Nagy, Roland; Depollier, Julien; Bucheton, Alain; Pelisson, Alain; Darlix, Jean-Luc

2006-01-01

Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNA(Lys,2) to two distinct primer binding sites (PBS) at the genome 5' and 3' ends. Only the 5' PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells.

Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

PubMed Central

Gabus, Caroline; Ivanyi-Nagy, Roland; Depollier, Julien; Bucheton, Alain; Pelisson, Alain; Darlix, Jean-Luc

2006-01-01

Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNALys,2 to two distinct primer binding sites (PBS) at the genome 5′ and 3′ ends. Only the 5′ PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells. PMID:17040893

Contribution of Mobile Group II Introns to Sinorhizobium meliloti Genome Evolution.

PubMed

Toro, Nicolás; Martínez-Abarca, Francisco; Molina-Sánchez, María D; García-Rodríguez, Fernando M; Nisa-Martínez, Rafael

2018-01-01

Mobile group II introns are ribozymes and retroelements that probably originate from bacteria. Sinorhizobium meliloti , the nitrogen-fixing endosymbiont of legumes of genus Medicago , harbors a large number of these retroelements. One of these elements, RmInt1, has been particularly successful at colonizing this multipartite genome. Many studies have improved our understanding of RmInt1 and phylogenetically related group II introns, their mobility mechanisms, spread and dynamics within S. meliloti and closely related species. Although RmInt1 conserves the ancient retroelement behavior, its evolutionary history suggests that this group II intron has played a role in the short- and long-term evolution of the S. meliloti genome. We will discuss its proposed role in genome evolution by controlling the spread and coexistence of potentially harmful mobile genetic elements, by ectopic transposition to different genetic loci as a source of early genomic variation and by generating sequence variation after a very slow degradation process, through intron remnants that may have continued to evolve, contributing to bacterial speciation.

Contribution of Mobile Group II Introns to Sinorhizobium meliloti Genome Evolution

PubMed Central

Toro, Nicolás; Martínez-Abarca, Francisco; Molina-Sánchez, María D.; García-Rodríguez, Fernando M.; Nisa-Martínez, Rafael

2018-01-01

Mobile group II introns are ribozymes and retroelements that probably originate from bacteria. Sinorhizobium meliloti, the nitrogen-fixing endosymbiont of legumes of genus Medicago, harbors a large number of these retroelements. One of these elements, RmInt1, has been particularly successful at colonizing this multipartite genome. Many studies have improved our understanding of RmInt1 and phylogenetically related group II introns, their mobility mechanisms, spread and dynamics within S. meliloti and closely related species. Although RmInt1 conserves the ancient retroelement behavior, its evolutionary history suggests that this group II intron has played a role in the short- and long-term evolution of the S. meliloti genome. We will discuss its proposed role in genome evolution by controlling the spread and coexistence of potentially harmful mobile genetic elements, by ectopic transposition to different genetic loci as a source of early genomic variation and by generating sequence variation after a very slow degradation process, through intron remnants that may have continued to evolve, contributing to bacterial speciation. PMID:29670598

Isolation and sequence analysis of the wheat B genome subtelomeric DNA.

PubMed

Salina, Elena A; Sergeeva, Ekaterina M; Adonina, Irina G; Shcherban, Andrey B; Afonnikov, Dmitry A; Belcram, Harry; Huneau, Cecile; Chalhoub, Boulos

2009-09-05

Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome) clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119,737 bp was annotated. It is composed of 33% transposable elements (TEs), 8.2% Spelt52 (namely, the subfamily Spelt52.2) and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11,666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags) suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0). Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat chromosomes. It has been demonstrated for the first time that Spelt52 sequences were involved in the evolution of terminal regions of common wheat chromosomes. Our research provides new insights into the microcollinearity in the terminal regions of wheat chromosomes 4BL and rice chromosome 3S.

Silencing of the PiAvr3a effector-encoding gene from Phytophthora infestans by transcriptional fusion to a short interspersed element.

PubMed

Vetukuri, Ramesh R; Tian, Zhendong; Avrova, Anna O; Savenkov, Eugene I; Dixelius, Christina; Whisson, Stephen C

2011-12-01

Phytophthora infestans is the notorious oomycete causing late blight of potato and tomato. A large proportion of the P. infestans genome is composed of transposable elements, the activity of which may be controlled by RNA silencing. Accumulation of small RNAs is one of the hallmarks of RNA silencing. Here we demonstrate the presence of small RNAs corresponding to the sequence of a short interspersed retrotransposable element (SINE) suggesting that small RNAs might be involved in silencing of SINEs in P. infestans. This notion was exploited to develop novel tools for gene silencing in P. infestans by engineering transcriptional fusions of the PiAvr3a gene, encoding an RXLR avirulence effector, to the infSINEm retroelement. Transgenic P. infestans lines expressing either 5'-infSINEm::PiAvr3a-3' or 5'-PiAvr3a::SINEm-3' chimeric transcripts initially exhibited partial silencing of PiAvr3a. Over time, PiAvr3a either recovered wild type transcript levels in some lines, or became fully silenced in others. Introduction of an inverted repeat construct was also successful in yielding P. infestans transgenic lines silenced for PiAvr3a. In contrast, constructs expressing antisense or aberrant RNA transcripts failed to initiate silencing of PiAvr3a. Lines exhibiting the most effective silencing of PiAvr3a were either weakly or non-pathogenic on susceptible potato cv. Bintje. This study expands the repertoire of reverse genetics tools available for P. infestans research, and provides insights into a possible mode of variation in effector expression through spread of silencing from adjacent retroelements. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Molecular reconstruction of extinct LINE-1 elements and their interaction with nonautonomous elements.

PubMed

Wagstaff, Bradley J; Kroutter, Emily N; Derbes, Rebecca S; Belancio, Victoria P; Roy-Engel, Astrid M

2013-01-01

Non-long terminal repeat retroelements continue to impact the human genome through cis-activity of long interspersed element-1 (LINE-1 or L1) and trans-mobilization of Alu. Current activity is dominated by modern subfamilies of these elements, leaving behind an evolutionary graveyard of extinct Alu and L1 subfamilies. Because Alu is a nonautonomous element that relies on L1 to retrotranspose, there is the possibility that competition between these elements has driven selection and antagonistic coevolution between Alu and L1. Through analysis of synonymous versus nonsynonymous codon evolution across L1 subfamilies, we find that the C-terminal ORF2 cys domain experienced a dramatic increase in amino acid substitution rate in the transition from L1PA5 to L1PA4 subfamilies. This observation coincides with the previously reported rapid evolution of ORF1 during the same transition period. Ancestral Alu sequences have been previously reconstructed, as their short size and ubiquity have made it relatively easy to retrieve consensus sequences from the human genome. In contrast, creating constructs of extinct L1 copies is a more laborious task. Here, we report our efforts to recreate and evaluate the retrotransposition capabilities of two ancestral L1 elements, L1PA4 and L1PA8 that were active ~18 and ~40 Ma, respectively. Relative to the modern L1PA1 subfamily, we find that both elements are similarly active in a cell culture retrotransposition assay in HeLa, and both are able to efficiently trans-mobilize Alu elements from several subfamilies. Although we observe some variation in Alu subfamily retrotransposition efficiency, any coevolution that may have occurred between LINEs and SINEs is not evident from these data. Population dynamics and stochastic variation in the number of active source elements likely play an important role in individual LINE or SINE subfamily amplification. If coevolution also contributes to changing retrotransposition rates and the progression of subfamilies, cell factors are likely to play an important mediating role in changing LINE-SINE interactions over evolutionary time.

Molecular Reconstruction of Extinct LINE-1 Elements and Their Interaction with Nonautonomous Elements

PubMed Central

Wagstaff, Bradley J.; Kroutter, Emily N.; Derbes, Rebecca S.; Belancio, Victoria P.; Roy-Engel, Astrid M.

2013-01-01

Non-long terminal repeat retroelements continue to impact the human genome through cis-activity of long interspersed element-1 (LINE-1 or L1) and trans-mobilization of Alu. Current activity is dominated by modern subfamilies of these elements, leaving behind an evolutionary graveyard of extinct Alu and L1 subfamilies. Because Alu is a nonautonomous element that relies on L1 to retrotranspose, there is the possibility that competition between these elements has driven selection and antagonistic coevolution between Alu and L1. Through analysis of synonymous versus nonsynonymous codon evolution across L1 subfamilies, we find that the C-terminal ORF2 cys domain experienced a dramatic increase in amino acid substitution rate in the transition from L1PA5 to L1PA4 subfamilies. This observation coincides with the previously reported rapid evolution of ORF1 during the same transition period. Ancestral Alu sequences have been previously reconstructed, as their short size and ubiquity have made it relatively easy to retrieve consensus sequences from the human genome. In contrast, creating constructs of extinct L1 copies is a more laborious task. Here, we report our efforts to recreate and evaluate the retrotransposition capabilities of two ancestral L1 elements, L1PA4 and L1PA8 that were active ∼18 and ∼40 Ma, respectively. Relative to the modern L1PA1 subfamily, we find that both elements are similarly active in a cell culture retrotransposition assay in HeLa, and both are able to efficiently trans-mobilize Alu elements from several subfamilies. Although we observe some variation in Alu subfamily retrotransposition efficiency, any coevolution that may have occurred between LINEs and SINEs is not evident from these data. Population dynamics and stochastic variation in the number of active source elements likely play an important role in individual LINE or SINE subfamily amplification. If coevolution also contributes to changing retrotransposition rates and the progression of subfamilies, cell factors are likely to play an important mediating role in changing LINE-SINE interactions over evolutionary time. PMID:22918960

The removal of RNA primers from DNA synthesized by the reverse transcriptase of the retrotransposon Tf1 is stimulated by Tf1 integrase.

PubMed

Herzig, Eytan; Voronin, Nickolay; Hizi, Amnon

2012-06-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process.

The Removal of RNA Primers from DNA Synthesized by the Reverse Transcriptase of the Retrotransposon Tf1 Is Stimulated by Tf1 Integrase

PubMed Central

Herzig, Eytan; Voronin, Nickolay

2012-01-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process. PMID:22491446

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes

PubMed Central

Rai, Sudhir Kumar; Sangesland, Maya; Lee, Michael; Esnault, Caroline; Cui, Yujin; Chatterjee, Atreyi Ghatak

2017-01-01

Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements. PMID:29232693

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes.

PubMed

Rai, Sudhir Kumar; Sangesland, Maya; Lee, Michael; Esnault, Caroline; Cui, Yujin; Chatterjee, Atreyi Ghatak; Levin, Henry L

2017-12-01

Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.

LTR Retrotransposons Show Low Levels of Unequal Recombination and High Rates of Intraelement Gene Conversion in Large Plant Genomes

PubMed Central

Cossu, Rosa Maria; Casola, Claudio; Giacomello, Stefania; Vidalis, Amaryllis

2017-01-01

Abstract The accumulation and removal of transposable elements (TEs) is a major driver of genome size evolution in eukaryotes. In plants, long terminal repeat (LTR) retrotransposons (LTR-RTs) represent the majority of TEs and form most of the nuclear DNA in large genomes. Unequal recombination (UR) between LTRs leads to removal of intervening sequence and formation of solo-LTRs. UR is a major mechanism of LTR-RT removal in many angiosperms, but our understanding of LTR-RT-associated recombination within the large, LTR-RT-rich genomes of conifers is quite limited. We employ a novel read-based methodology to estimate the relative rates of LTR-RT-associated UR within the genomes of four conifer and seven angiosperm species. We found the lowest rates of UR in the largest genomes studied, conifers and the angiosperm maize. Recombination may also resolve as gene conversion, which does not remove sequence, so we analyzed LTR-RT-associated gene conversion events (GCEs) in Norway spruce and six angiosperms. Opposite the trend for UR, we found the highest rates of GCEs in Norway spruce and maize. Unlike previous work in angiosperms, we found no evidence that rates of UR correlate with retroelement structural features in the conifers, suggesting that another process is suppressing UR in these species. Recent results from diverse eukaryotes indicate that heterochromatin affects the resolution of recombination, by favoring gene conversion over crossing-over, similar to our observation of opposed rates of UR and GCEs. Control of LTR-RT proliferation via formation of heterochromatin would be a likely step toward large genomes in eukaryotes carrying high LTR-RT content. PMID:29228262

Nonneutral GC3 and retroelement codon mimicry in Phytophthora.

PubMed

Jiang, Rays H Y; Govers, Francine

2006-10-01

Phytophthora is a genus entirely comprised of destructive plant pathogens. It belongs to the Stramenopila, a unique branch of eukaryotes, phylogenetically distinct from plants, animals, or fungi. Phytophthora genes show a strong preference for usage of codons ending with G or C (high GC3). The presence of high GC3 in genes can be utilized to differentiate coding regions from noncoding regions in the genome. We found that both selective pressure and mutation bias drive codon bias in Phytophthora. Indicative for selection pressure is the higher GC3 value of highly expressed genes in different Phytophthora species. Lineage specific GC increase of noncoding regions is reminiscent of whole-genome mutation bias, whereas the elevated Phytophthora GC3 is primarily a result of translation efficiency-driven selection. Heterogeneous retrotransposons exist in Phytophthora genomes and many of them vary in their GC content. Interestingly, the most widespread groups of retroelements in Phytophthora show high GC3 and a codon bias that is similar to host genes. Apparently, selection pressure has been exerted on the retroelement's codon usage, and such mimicry of host codon bias might be beneficial for the propagation of retrotransposons.

Compact genome of the Antarctic midge is likely an adaptation to an extreme environment.

PubMed

Kelley, Joanna L; Peyton, Justin T; Fiston-Lavier, Anna-Sophie; Teets, Nicholas M; Yee, Muh-Ching; Johnston, J Spencer; Bustamante, Carlos D; Lee, Richard E; Denlinger, David L

2014-08-12

The midge, Belgica antarctica, is the only insect endemic to Antarctica, and thus it offers a powerful model for probing responses to extreme temperatures, freeze tolerance, dehydration, osmotic stress, ultraviolet radiation and other forms of environmental stress. Here we present the first genome assembly of an extremophile, the first dipteran in the family Chironomidae, and the first Antarctic eukaryote to be sequenced. At 99 megabases, B. antarctica has the smallest insect genome sequenced thus far. Although it has a similar number of genes as other Diptera, the midge genome has very low repeat density and a reduction in intron length. Environmental extremes appear to constrain genome architecture, not gene content. The few transposable elements present are mainly ancient, inactive retroelements. An abundance of genes associated with development, regulation of metabolism and responses to external stimuli may reflect adaptations for surviving in this harsh environment.

Compact genome of the Antarctic midge is likely an adaptation to an extreme environment

PubMed Central

Kelley, Joanna L.; Peyton, Justin T.; Fiston-Lavier, Anna-Sophie; Teets, Nicholas M.; Yee, Muh-Ching; Johnston, J. Spencer; Bustamante, Carlos D.; Lee, Richard E.; Denlinger, David L.

2014-01-01

The midge, Belgica antarctica, is the only insect endemic to Antarctica, and thus it offers a powerful model for probing responses to extreme temperatures, freeze tolerance, dehydration, osmotic stress, ultraviolet radiation and other forms of environmental stress. Here we present the first genome assembly of an extremophile, the first dipteran in the family Chironomidae, and the first Antarctic eukaryote to be sequenced. At 99 megabases, B. antarctica has the smallest insect genome sequenced thus far. Although it has a similar number of genes as other Diptera, the midge genome has very low repeat density and a reduction in intron length. Environmental extremes appear to constrain genome architecture, not gene content. The few transposable elements present are mainly ancient, inactive retroelements. An abundance of genes associated with development, regulation of metabolism and responses to external stimuli may reflect adaptations for surviving in this harsh environment. PMID:25118180

Retinoic acid-induced differentiation of retrovirus-infected HL-60 cells is associated with enhanced transcription from the viral long terminal repeat

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

Collins, S.J.

1988-11-01

The author infected different human leukemic cell lines with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. In retrovirus-infected G418-resistant HL-60 cells, induction of granulocyte differentiation by retinoic acid was invariably accompanied by a marked increase (5- to 10-fold) in the transcriptional activity of the integrated retroviral long terminal repeat.

Centromere and telomere sequence alterations reflect the rapid genome evolution within the carnivorous plant genus Genlisea.

PubMed

Tran, Trung D; Cao, Hieu X; Jovtchev, Gabriele; Neumann, Pavel; Novák, Petr; Fojtová, Miloslava; Vu, Giang T H; Macas, Jiří; Fajkus, Jiří; Schubert, Ingo; Fuchs, Joerg

2015-12-01

Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra displayed a combination of four retroelements at centromeric positions. G. nigrocaulis and G. pygmaea chromosome ends are characterized by the Arabidopsis-type telomeric repeats (TTTAGGG); G. hispidula and G. subglabra instead revealed two intermingled sequence variants (TTCAGG and TTTCAGG). These differences in centromeric and, surprisingly, also in telomeric DNA sequences, uncovered between groups with on average a > 9-fold genome size difference, emphasize the fast genome evolution within this genus. Such intrageneric evolutionary alteration of telomeric repeats with cytosine in the guanine-rich strand, not yet known for plants, might impact the epigenetic telomere chromatin modification. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing.

PubMed

Hribová, Eva; Neumann, Pavel; Matsumoto, Takashi; Roux, Nicolas; Macas, Jirí; Dolezel, Jaroslav

2010-09-16

Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing

PubMed Central

2010-01-01

Background Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. Results In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. Conclusion A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection. PMID:20846365

The RNA gene information: retroelement-microRNA entangling as the RNA quantum code.

PubMed

Fujii, Yoichi Robertus

2013-01-01

MicroRNA (miRNA) and retroelements may be a master of regulator in our life, which are evolutionally involved in the origin of species. To support the Darwinism from the aspect of molecular evolution process, it has tremendously been interested in the molecular information of naive RNA. The RNA wave model 2000 consists of four concepts that have altered from original idea of the miRNA genes for crosstalk among embryonic stem cells, their niche cells, and retroelements as a carrier vesicle of the RNA genes. (1) the miRNA gene as a mobile genetic element induces transcriptional and posttranscriptional silencing via networking-processes (no hierarchical architecture); (2) the RNA information supplied by the miRNA genes expands to intracellular, intercellular, intraorgan, interorgan, intraspecies, and interspecies under the cycle of life into the global environment; (3) the mobile miRNAs can self-proliferate; and (4) cells contain two types information as resident and genomic miRNAs. Based on RNA wave, we have developed an interest in investigation of the transformation from RNA information to quantum bits as physicochemical characters of RNA with the measurement of RNA electron spin. When it would have been given that the fundamental bases for the acquired characters in genetics can be controlled by RNA gene information, it may be available to apply for challenging against RNA gene diseases, such as stress-induced diseases.

LINE-1 silencing by retinoblastoma proteins is effected through the nucleosomal and remodeling deacetylase multiprotein complex.

PubMed

Montoya-Durango, Diego E; Ramos, Kenneth A; Bojang, Pasano; Ruiz, Lorell; Ramos, Irma N; Ramos, Kenneth S

2016-01-25

Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells. Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5'-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-β, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-β, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells. RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function.

Genomic patterns associated with paternal/maternal distribution of transposable elements

NASA Astrophysics Data System (ADS)

Jurka, Jerzy

2003-03-01

Transposable elements (TEs) are specialized DNA or RNA fragments capable of surviving in intragenomic niches. They are commonly, perhaps unjustifiably referred to as "selfish" or "parasitic" elements. TEs can be divided in two major classes: retroelements and DNA transposons. The former include non-LTR retrotransposons and retrovirus-like elements, using reverse transriptase for their reproduction prior to integration into host DNA. The latter depend mostly on host DNA replication, with possible exception of rolling-circle transposons recently discovered by our team. I will review basic information on TEs, with emphasis on human Alu and L1 retroelements discussed in the context of genomic organization. TEs are non-randomly distributed in chromosomal DNA. In particular, human Alu elements tend to prefer GC-rich regions, whereas L1 accumulate in AT-rich regions. Current explanations of this phenomenon focus on the so called "target effects" and post-insertional selection. However, the proposed models appear to be unsatisfactory and alternative explanations invoking "channeling" to different chromosomal regions will be a major focus of my presentation. Transposable elements (TEs) can be expressed and integrated into host DNA in the male or female germlines, or both. Different models of expression and integration imply different proportions of TEs on sex chromosomes and autosomes. The density of recently retroposed human Alu elements is around three times higher on chromosome Y than on chromosome X, and over two times higher than the average density for all human autosomes. This implies Alu activity in paternal germlines. Analogous inter-chromosomal proportions for other repeat families should determine their compatibility with one of the three basic models describing the inheritance of TEs. Published evidence indicates that maternally and paternally imprinted genes roughly correspond to GC-rich and AT-rich DNA. This may explain the observed chromosomal distribution of Alu and L1 elements. Finally, paternal models of inheritance predict rapid accumulation of active TEs on chromosome Y. I will discuss potential implications of this phenomenon for evolution of chromosome Y and transposable elements.

Insertion of reticuloendotheliosis virus long terminal repeat into CVI988 strain of Marek’s disease virus results in enhanced growth and protection

USDA-ARS?s Scientific Manuscript database

It has been reported that co-cultivation of a JM/102W strain, a virulent strain of Marek’s disease virus (MDV), with reticuloendotheliosis virus (REV) resulted in the integration of REV long terminal repeat (LTR) into the MDV repeat region. The resulting virus, RM1, was unable to transform T-cells ...

Repetitive DNA and Plant Domestication: Variation in Copy Number and Proximity to Genes of LTR-Retrotransposons among Wild and Cultivated Sunflower (Helianthus annuus) Genotypes

PubMed Central

Mascagni, Flavia; Barghini, Elena; Giordani, Tommaso; Rieseberg, Loren H.; Cavallini, Andrea; Natali, Lucia

2015-01-01

The sunflower (Helianthus annuus) genome contains a very large proportion of transposable elements, especially long terminal repeat retrotransposons. However, knowledge on the retrotransposon-related variability within this species is still limited. We used next-generation sequencing (NGS) technologies to perform a quantitative and qualitative survey of intraspecific variation of the retrotransposon fraction of the genome across 15 genotypes—7 wild accessions and 8 cultivars—of H. annuus. By mapping the Illumina reads of the 15 genotypes onto a library of sunflower long terminal repeat retrotransposons, we observed considerable variability in redundancy among genotypes, at both superfamily and family levels. In another analysis, we mapped Illumina paired reads to two sets of sequences, that is, long terminal repeat retrotransposons and protein-encoding sequences, and evaluated the extent of retrotransposon proximity to genes in the sunflower genome by counting the number of paired reads in which one read mapped to a retrotransposon and the other to a gene. Large variability among genotypes was also ascertained for retrotransposon proximity to genes. Both long terminal repeat retrotransposon redundancy and proximity to genes varied among retrotransposon families and also between cultivated and wild genotypes. Such differences are discussed in relation to the possible role of long terminal repeat retrotransposons in the domestication of sunflower. PMID:26608057

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

PubMed

Pritham, Ellen J; Putliwala, Tasneem; Feschotte, Cédric

2007-04-01

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.

RNA-Seq Analysis to Measure the Expression of SINE Retroelements.

PubMed

Román, Ángel Carlos; Morales-Hernández, Antonio; Fernández-Salguero, Pedro M

2016-01-01

The intrinsic features of retroelements, like their repetitive nature and disseminated presence in their host genomes, demand the use of advanced methodologies for their bioinformatic and functional study. The short length of SINE (short interspersed elements) retrotransposons makes such analyses even more complex. Next-generation sequencing (NGS) technologies are currently one of the most widely used tools to characterize the whole repertoire of gene expression in a specific tissue. In this chapter, we will review the molecular and computational methods needed to perform NGS analyses on SINE elements. We will also describe new methods of potential interest for researchers studying repetitive elements. We intend to outline the general ideas behind the computational analyses of NGS data obtained from SINE elements, and to stimulate other scientists to expand our current knowledge on SINE biology using RNA-seq and other NGS tools.

Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

PubMed Central

Hajek, Kathryn L.; Friesen, Paul D.

1998-01-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging. PMID:9765414

Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family.

PubMed

Hajek, K L; Friesen, P D

1998-11-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

Bacterial Group II Introns: Identification and Mobility Assay.

PubMed

Toro, Nicolás; Molina-Sánchez, María Dolores; Nisa-Martínez, Rafael; Martínez-Abarca, Francisco; García-Rodríguez, Fernando Manuel

2016-01-01

Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.

Repetitive DNA and Plant Domestication: Variation in Copy Number and Proximity to Genes of LTR-Retrotransposons among Wild and Cultivated Sunflower (Helianthus annuus) Genotypes.

PubMed

Mascagni, Flavia; Barghini, Elena; Giordani, Tommaso; Rieseberg, Loren H; Cavallini, Andrea; Natali, Lucia

2015-11-24

The sunflower (Helianthus annuus) genome contains a very large proportion of transposable elements, especially long terminal repeat retrotransposons. However, knowledge on the retrotransposon-related variability within this species is still limited. We used next-generation sequencing (NGS) technologies to perform a quantitative and qualitative survey of intraspecific variation of the retrotransposon fraction of the genome across 15 genotypes--7 wild accessions and 8 cultivars--of H. annuus. By mapping the Illumina reads of the 15 genotypes onto a library of sunflower long terminal repeat retrotransposons, we observed considerable variability in redundancy among genotypes, at both superfamily and family levels. In another analysis, we mapped Illumina paired reads to two sets of sequences, that is, long terminal repeat retrotransposons and protein-encoding sequences, and evaluated the extent of retrotransposon proximity to genes in the sunflower genome by counting the number of paired reads in which one read mapped to a retrotransposon and the other to a gene. Large variability among genotypes was also ascertained for retrotransposon proximity to genes. Both long terminal repeat retrotransposon redundancy and proximity to genes varied among retrotransposon families and also between cultivated and wild genotypes. Such differences are discussed in relation to the possible role of long terminal repeat retrotransposons in the domestication of sunflower. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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

Zweifel,M.; Leahy, D.; Barrick, D.

Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. Here we report the structure of the Drosophila Deltex N-terminal domain, which contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal tomore » the second WWE domain. We demonstrate that although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination.« less

Retrotransposons Control Fruit-Specific, Cold-Dependent Accumulation of Anthocyanins in Blood Oranges[W][OA

PubMed Central

Butelli, Eugenio; Licciardello, Concetta; Zhang, Yang; Liu, Jianjun; Mackay, Steve; Bailey, Paul; Reforgiato-Recupero, Giuseppe; Martin, Cathie

2012-01-01

Traditionally, Sicilian blood oranges (Citrus sinensis) have been associated with cardiovascular health, and consumption has been shown to prevent obesity in mice fed a high-fat diet. Despite increasing consumer interest in these health-promoting attributes, production of blood oranges remains unreliable due largely to a dependency on cold for full color formation. We show that Sicilian blood orange arose by insertion of a Copia-like retrotransposon adjacent to a gene encoding Ruby, a MYB transcriptional activator of anthocyanin production. The retrotransposon controls Ruby expression, and cold dependency reflects the induction of the retroelement by stress. A blood orange of Chinese origin results from an independent insertion of a similar retrotransposon, and color formation in its fruit is also cold dependent. Our results suggest that transposition and recombination of retroelements are likely important sources of variation in Citrus. PMID:22427337

Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington’s disease

PubMed Central

Chen, Mingchen; Wolynes, Peter G.

2017-01-01

Huntington’s disease (HD) is a neurodegenerative disease caused by an abnormal expansion in the polyglutamine (polyQ) track of the Huntingtin (HTT) protein. The severity of the disease depends on the polyQ repeat length, arising only in patients with proteins having 36 repeats or more. Previous studies have shown that the aggregation of N-terminal fragments (encoded by HTT exon 1) underlies the disease pathology in mouse models and that the HTT exon 1 gene product can self-assemble into amyloid structures. Here, we provide detailed structural mechanisms for aggregation of several protein fragments encoded by HTT exon 1 by using the associative memory, water-mediated, structure and energy model (AWSEM) to construct their free energy landscapes. We find that the addition of the N-terminal 17-residue sequence (NT17) facilitates polyQ aggregation by encouraging the formation of prefibrillar oligomers, whereas adding the C-terminal polyproline sequence (P10) inhibits aggregation. The combination of both terminal additions in HTT exon 1 fragment leads to a complex aggregation mechanism with a basic core that resembles that found for the aggregation of pure polyQ repeats using AWSEM. At the extrapolated physiological concentration, although the grand canonical free energy profiles are uphill for HTT exon 1 fragments having 20 or 30 glutamines, the aggregation landscape for fragments with 40 repeats has become downhill. This computational prediction agrees with the critical length found for the onset of HD and suggests potential therapies based on blocking early binding events involving the terminal additions to the polyQ repeats. PMID:28400517

Dynamic probability of reinforcement for cooperation: Random game termination in the centipede game.

PubMed

Krockow, Eva M; Colman, Andrew M; Pulford, Briony D

2018-03-01

Experimental games have previously been used to study principles of human interaction. Many such games are characterized by iterated or repeated designs that model dynamic relationships, including reciprocal cooperation. To enable the study of infinite game repetitions and to avoid endgame effects of lower cooperation toward the final game round, investigators have introduced random termination rules. This study extends previous research that has focused narrowly on repeated Prisoner's Dilemma games by conducting a controlled experiment of two-player, random termination Centipede games involving probabilistic reinforcement and characterized by the longest decision sequences reported in the empirical literature to date (24 decision nodes). Specifically, we assessed mean exit points and cooperation rates, and compared the effects of four different termination rules: no random game termination, random game termination with constant termination probability, random game termination with increasing termination probability, and random game termination with decreasing termination probability. We found that although mean exit points were lower for games with shorter expected game lengths, the subjects' cooperativeness was significantly reduced only in the most extreme condition with decreasing computer termination probability and an expected game length of two decision nodes. © 2018 Society for the Experimental Analysis of Behavior.

25 CFR 11.1114 - Termination.

Code of Federal Regulations, 2010 CFR

2010-04-01

... functions; (iii) The parent(s) has subjected the minor to willful and repeated acts of sexual abuse; (iv... Minor-in-Need-of-Care Procedure § 11.1114 Termination. (a) Parental rights to a child may be terminated by the children's court according to the procedures in this section. (b) Proceedings to terminate...

Genetic Studies of the Prp17 Gene of Saccharomyces Cerevisiae: A Domain Essential for Function Maps to a Nonconserved Region of the Protein

PubMed Central

Seshadri, V.; Vaidya, V. C.; Vijayraghavan, U.

1996-01-01

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the β transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the β transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is in the N terminal region of the protein. PMID:8722761

Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

USDA-ARS?s Scientific Manuscript database

Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insec...

Variation, Repetition, And Choice

PubMed Central

Abreu-Rodrigues, Josele; Lattal, Kennon A; dos Santos, Cristiano V; Matos, Ricardo A

2005-01-01

Experiment 1 investigated the controlling properties of variability contingencies on choice between repeated and variable responding. Pigeons were exposed to concurrent-chains schedules with two alternatives. In the REPEAT alternative, reinforcers in the terminal link depended on a single sequence of four responses. In the VARY alternative, a response sequence in the terminal link was reinforced only if it differed from the n previous sequences (lag criterion). The REPEAT contingency generated low, constant levels of sequence variation whereas the VARY contingency produced levels of sequence variation that increased with the lag criterion. Preference for the REPEAT alternative tended to increase directly with the degree of variation required for reinforcement. Experiment 2 examined the potential confounding effects in Experiment 1 of immediacy of reinforcement by yoking the interreinforcer intervals in the REPEAT alternative to those in the VARY alternative. Again, preference for REPEAT was a function of the lag criterion. Choice between varying and repeating behavior is discussed with respect to obtained behavioral variability, probability of reinforcement, delay of reinforcement, and switching within a sequence. PMID:15828592

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

PubMed

Sharma, Upasna; Conine, Colin C; Shea, Jeremy M; Boskovic, Ana; Derr, Alan G; Bing, Xin Y; Belleannee, Clemence; Kucukural, Alper; Serra, Ryan W; Sun, Fengyun; Song, Lina; Carone, Benjamin R; Ricci, Emiliano P; Li, Xin Z; Fauquier, Lucas; Moore, Melissa J; Sullivan, Robert; Mello, Craig C; Garber, Manuel; Rando, Oliver J

2016-01-22

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo. Copyright © 2016, American Association for the Advancement of Science.

Traceability of Asian Matsutake, Specialty Mushrooms Produced by the Ectomycorrhizal Basidiomycete Tricholoma matsutake, on the Basis of Retroelement-Based DNA Markers▿

PubMed Central

Murata, Hitoshi; Babasaki, Katsuhiko; Saegusa, Tomoki; Takemoto, Kenji; Yamada, Akiyoshi; Ohta, Akira

2008-01-01

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies, matsutake, in forests. Here we report a PCR system targeting retroelement integration sites to differentiate among individual Asian isolates of T. matsutake based on their geographical origins, such as Japan, the area of South Korea through North Korea, the northeastern provinces of China, and the area of the southwestern provinces of China through Bhutan. The overall misjudgment rate of the analytical system was approximately 5% based on 95 samples of T. matsutake examined including those from cultures and from commodities. We also provide evidence that T. matsutake isolates grown throughout the Far East, including the northeastern provinces of China, are closely related to each other while distinct from those in the area of the southwestern provinces of China through Bhutan. The method allows us to trace back geographical origins of Asian matsutake, thus contributing to food safety, appropriate tariffs, and proper price setting. PMID:18281433

The APOBEC3 Family of Retroelement Restriction Factors

PubMed Central

Refsland, Eric W.; Harris, Reuben S.

2014-01-01

The ability to regulate and even target mutagenesis is an extremely valuable cellular asset. Enzyme-catalyzed DNA cytosine deamination is a molecular strategy employed by vertebrates to promote antibody diversity and defend against foreign nucleic acids. Ten years ago, a family of cellular enzymes was first described with several proving capable of deaminating DNA and inhibiting HIV-1 replication. Ensuing studies on the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) restriction factors have uncovered a broad-spectrum innate defense network that suppresses the replication of numerous endogenous and exogenous DNA-based parasites. Although many viruses possess equally elaborate counter-defense mechanisms, the APOBEC3 enzymes offer a tantalizing possibility of leveraging innate immunity to fend off viral infection. Here we focus on mechanisms of retroelement restriction by the APOBEC3 family of restriction enzymes and we consider the therapeutic benefits, as well as the possible pathological consequences, of arming cells with active DNA deaminases. PMID:23686230

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors*

PubMed Central

Chong, Huihui; Yao, Xue; Sun, Jianping; Qiu, Zonglin; Zhang, Meng; Waltersperger, Sandro; Wang, Meitian; Cui, Sheng; He, Yuxian

2012-01-01

CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors. PMID:22879603

Amino terminus of substance P potentiates kainic acid-induced activity in the mouse spinal cord.

PubMed

Larson, A A; Sun, X

1992-12-01

Sensitization to the behavioral effects produced by repeated injections of kainic acid (KA) into the mouse spinal cord area has been previously shown to be abolished by pretreatment with capsaicin, a neurotoxin of substance P (SP)-containing primary afferent C-fibers. While SP has a variety of well characterized biological actions that are mediated by interactions of its COOH terminus with neurokinin receptors, more recently we have characterized an amino-terminally directed SP binding site. The present studies were initiated to determine whether behavioral sensitization to repeated injections of intrathecally administered KA is mediated by the COOH or NH2 terminal of SP. In the present studies, pretreatment with SP(1-7), an NH2-terminal fragment of SP, but not SP(5-11), a COOH-terminal fragment, potentiated KA-induced behavioral activity in mice. Pretreatment with [D-Pro2,D-Phe7]SP(1-7), an inhibitor of SP NH2-terminal binding, blocked the potentiative effect of SP(1-7) as well as the sensitization to repeated injections of KA. In contrast, [D-Pro2,D-Trp7,9]SP, a neurokinin antagonist, had little effect on behavioral sensitization to KA. The present study suggests that SP has an important modulatory role on excitatory amino acid activity in the spinal cord that is mediated by an action of the NH2 terminal of SP at a non-neurokinin receptor.

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

PubMed Central

Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

2014-01-01

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

Terminal-Repeat Retrotransposons with GAG Domain in Plant Genomes: A New Testimony on the Complex World of Transposable Elements

PubMed Central

Chaparro, Cristian; Gayraud, Thomas; de Souza, Rogerio Fernandes; Domingues, Douglas Silva; Akaffou, Sélastique; Laforga Vanzela, Andre Luis; de Kochko, Alexandre; Rigoreau, Michel; Crouzillat, Dominique; Hamon, Serge; Hamon, Perla; Guyot, Romain

2015-01-01

A novel structure of nonautonomous long terminal repeat (LTR) retrotransposons called terminal repeat with GAG domain (TR-GAG) has been described in plants, both in monocotyledonous, dicotyledonous and basal angiosperm genomes. TR-GAGs are relatively short elements in length (<4 kb) showing the typical features of LTR-retrotransposons. However, they carry only one open reading frame coding for the GAG precursor protein involved for instance in transposition, the assembly, and the packaging of the element into the virus-like particle. GAG precursors show similarities with both Copia and Gypsy GAG proteins, suggesting evolutionary relationships of TR-GAG elements with both families. Despite the lack of the enzymatic machinery required for their mobility, strong evidences suggest that TR-GAGs are still active. TR-GAGs represent ubiquitous nonautonomous structures that could be involved in the molecular diversities of plant genomes. PMID:25573958

Novel surface attachment mechanism of the Streptococcus pneumoniae protein PspA.

PubMed Central

Yother, J; White, J M

1994-01-01

Pneumococcal surface protein A (PspA) of Streptococcus pneumoniae has been found to utilize a novel mechanism for anchoring to the bacterial cell surface. In contrast to that of surface proteins from other gram-positive bacteria, PspA anchoring required choline-mediated interactions between the membrane-associated lipoteichoic acid and the C-terminal repeat region of PspA. Release of PspA from the cell surface could be effected by deletion of 5 of the 10 C-terminal repeat units, by high concentrations of choline, or by growth in choline-deficient medium. Other pneumococcal proteins, including autolysin, which has a similar C-terminal repeat region, were not released by these treatments. The attachment mechanism utilized by PspA thus appears to be uniquely adapted to exploit the unusual structure of the pneumococcal cell surface. Further, it has provided the means for rapid and simple isolation of immunogenic PspA from S. pneumoniae. Images PMID:7910604

The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat.

PubMed Central

Lu, X; Welsh, T M; Peterlin, B M

1993-01-01

The human immunodeficiency virus type 1 long terminal repeat sets up two different transcription complexes, which have been called processive and nonprocessive complexes. By mutating and substituting cis-acting sequences, we mapped elements of the human immunodeficiency virus long terminal repeat that are responsible for creating each transcription complex. Whereas processive complexes are efficiently assembled by upstream promoter elements in the absence of the TATA box, nonprocessive complexes absolutely require the TATA box. Moreover, the TATA box alone can set up these nonprocessive complexes, and nonprocessive but not processive complexes are trans activated by Tat. Finally, a strong DNA-binding site between the TATA box and trans-activation-responsive region interferes with either the assembly or movement of these nonprocessive complexes and diminishes the effects of Tat. Thus, Tat affects a critical step in the formation of elongation-competent transcription complexes. Images PMID:8445708

Comparison of the carboxy-terminal DP-repeat region in the co-chaperones Hop and Hip

PubMed Central

Nelson, Gregory M.; Huffman, Holly; Smith, David F.

2003-01-01

Functional steroid receptor complexes are assembled and maintained by an ordered pathway of interactions involving multiple components of the cellular chaperone machinery. Two of these components, Hop and Hip, serve as co-chaperones to the major heat shock proteins (Hsps), Hsp70 and Hsp90, and participate in intermediate stages of receptor assembly. In an effort to better understand the functions of Hop and Hip in the assembly process, we focused on a region of similarity located near the C-terminus of each co-chaperone. Contained within this region is a repeated sequence motif we have termed the DP repeat. Earlier mutagenesis studies implicated the DP repeat of either Hop or Hip in Hsp70 binding and in normal assembly of the co-chaperones with progesterone receptor (PR) complexes. We report here that the DP repeat lies within a protease-resistant domain that extends to or is near the C-terminus of both co-chaperones. Point mutations in the DP repeats render the C-terminal regions hypersensitive to proteolysis. In addition, a Hop DP mutant displays altered proteolytic digestion patterns, which suggest that the DP-repeat region influences the folding of other Hop domains. Although the respective DP regions of Hop and Hip share sequence and structural similarities, they are not functionally interchangeable. Moreover, a double-point mutation within the second DP-repeat unit of Hop that converts this to the sequence found in Hip disrupts Hop function; however, the corresponding mutation in Hip does not alter its function. We conclude that the DP repeats are important structural elements within a C-terminal domain, which is important for Hop and Hip function. PMID:14627198

Comparison of the carboxy-terminal DP-repeat region in the co-chaperones Hop and Hip.

PubMed

Nelson, Gregory M; Huffman, Holly; Smith, David F

2003-01-01

Functional steroid receptor complexes are assembled and maintained by an ordered pathway of interactions involving multiple components of the cellular chaperone machinery. Two of these components, Hop and Hip, serve as co-chaperones to the major heat shock proteins (Hsps), Hsp70 and Hsp90, and participate in intermediate stages of receptor assembly. In an effort to better understand the functions of Hop and Hip in the assembly process, we focused on a region of similarity located near the C-terminus of each co-chaperone. Contained within this region is a repeated sequence motif we have termed the DP repeat. Earlier mutagenesis studies implicated the DP repeat of either Hop or Hip in Hsp70 binding and in normal assembly of the co-chaperones with progesterone receptor (PR) complexes. We report here that the DP repeat lies within a protease-resistant domain that extends to or is near the C-terminus of both co-chaperones. Point mutations in the DP repeats render the C-terminal regions hypersensitive to proteolysis. In addition, a Hop DP mutant displays altered proteolytic digestion patterns, which suggest that the DP-repeat region influences the folding of other Hop domains. Although the respective DP regions of Hop and Hip share sequence and structural similarities, they are not functionally interchangeable. Moreover, a double-point mutation within the second DP-repeat unit of Hop that converts this to the sequence found in Hip disrupts Hop function; however, the corresponding mutation in Hip does not alter its function. We conclude that the DP repeats are important structural elements within a C-terminal domain, which is important for Hop and Hip function.

The open reading frames in the 3' long terminal repeats of several mouse mammary tumor virus integrants encode V beta 3-specific superantigens

PubMed Central

1992-01-01

Mice expressing the minor lymphocyte stimulation antigens, Mls-1a, -2a, or -3a, singly on the B10.BR background have been generated. Mls phenotypes correlate with the integration of mouse mammary tumor viruses (MTV) in the mouse genome. The open reading frames within the 3' long terminal repeats of the integrated MTVs 1, 3, 6, and 13 encode V beta 3-specific superantigens. Sequence data for these viral superantigens is presented, indicating that it is the COOH-terminal portion of the viral superantigen that interacts with the T cell receptor V beta element. PMID:1309854

The C-terminal region of Ge-1 presents conserved structural features required for P-body localization.

PubMed

Jinek, Martin; Eulalio, Ana; Lingel, Andreas; Helms, Sigrun; Conti, Elena; Izaurralde, Elisa

2008-10-01

The removal of the 5' cap structure by the DCP1-DCP2 decapping complex irreversibly commits eukaryotic mRNAs to degradation. In human cells, the interaction between DCP1 and DCP2 is bridged by the Ge-1 protein. Ge-1 contains an N-terminal WD40-repeat domain connected by a low-complexity region to a conserved C-terminal domain. It was reported that the C-terminal domain interacts with DCP2 and mediates Ge-1 oligomerization and P-body localization. To understand the molecular basis for these functions, we determined the three-dimensional crystal structure of the most conserved region of the Drosophila melanogaster Ge-1 C-terminal domain. The region adopts an all alpha-helical fold related to ARM- and HEAT-repeat proteins. Using structure-based mutants we identified an invariant surface residue affecting P-body localization. The conservation of critical surface and structural residues suggests that the C-terminal region adopts a similar fold with conserved functions in all members of the Ge-1 protein family.

Structure and stability of the ankyrin domain of the Drosophila Notch receptor.

PubMed

Zweifel, Mark E; Leahy, Daniel J; Hughson, Frederick M; Barrick, Doug

2003-11-01

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

Multiple bidirectional initiations and terminations of transcription in the Marek's disease virus long repeat regions.

PubMed Central

Chen, X B; Velicer, L F

1991-01-01

Marek's disease is an oncogenic disease of chickens caused by a herpesvirus, Marek's disease virus (MDV). Serial in vitro passage of pathogenic MDV results in amplification of a 132-bp direct repeat in the MDV genome's TRL and IRL repeat regions and loss of tumorigenicity. This led to the hypothesis that upon such expansion, one or more tumor-inducing genes fail to be expressed. In this report a group of cDNAs mapping in the expanded regions were isolated from a pathogenic MDV strain in which the 132-bp direct repeat number was found to range between one and seven. Partial cDNA sequencing and S1 nuclease protection analysis revealed that the corresponding transcripts are either initiated or terminated within or near the expanded regions at multiple sites in both rightward and leftward directions. Furthermore, each 132-bp repeat contains one TATA box and two polyadenylation consensus sequences in each direction. These RNAs contain a partial copy or one or more full copies of the 132-bp direct repeat at either their 5' or 3' end. Northern (RNA) blot analysis showed that the majority of transcripts are 1.8 kb in size, while the minor species range in size from 0.67 to 3.1 kb. Together, these data raise the possibility that the 132-bp direct repeat, and indirectly its copy number, may be involved in the regulation of transcriptional initiation and termination and therefore in the generation of four groups of transcripts from the TRL and IRL, although this remains to be demonstrated. Images PMID:1850022

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis.

PubMed

von Bergen, Martin; Barghorn, Stefan; Müller, Shirley A; Pickhardt, Marcus; Biernat, Jacek; Mandelkow, Eva-Maria; Davies, Peter; Aebi, Ueli; Mandelkow, Eckhard

2006-05-23

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.

Passenger comfort during terminal-area flight maneuvers. M.S. Thesis.

NASA Technical Reports Server (NTRS)

Schoonover, W. E., Jr.

1976-01-01

A series of flight experiments was conducted to obtain passenger subjective responses to closely controlled and repeatable flight maneuvers. In 8 test flights, reactions were obtained from 30 passenger subjects to a wide range of terminal-area maneuvers, including descents, turns, decelerations, and combinations thereof. Analysis of the passenger rating variance indicated that the objective of a repeatable flight passenger environment was achieved. Multiple linear regression models developed from the test data were used to define maneuver motion boundaries for specified degrees of passenger acceptance.

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

The primary transcriptome of the marine diazotroph Trichodesmium erythraeum IMS101

NASA Astrophysics Data System (ADS)

Pfreundt, Ulrike; Kopf, Matthias; Belkin, Natalia; Berman-Frank, Ilana; Hess, Wolfgang R.

2014-08-01

Blooms of the dinitrogen-fixing marine cyanobacterium Trichodesmium considerably contribute to new nitrogen inputs into tropical oceans. Intriguingly, only 60% of the Trichodesmium erythraeum IMS101 genome sequence codes for protein, compared with ~85% in other sequenced cyanobacterial genomes. The extensive non-coding genome fraction suggests space for an unusually high number of unidentified, potentially regulatory non-protein-coding RNAs (ncRNAs). To identify the transcribed fraction of the genome, here we present a genome-wide map of transcriptional start sites (TSS) at single nucleotide resolution, revealing the activity of 6,080 promoters. We demonstrate that T. erythraeum has the highest number of actively splicing group II introns and the highest percentage of TSS yielding ncRNAs of any bacterium examined to date. We identified a highly transcribed retroelement that serves as template repeat for the targeted mutation of at least 12 different genes by mutagenic homing. Our findings explain the non-coding portion of the T. erythraeum genome by the transcription of an unusually high number of non-coding transcripts in addition to the known high incidence of transposable elements. We conclude that riboregulation and RNA maturation-dependent processes constitute a major part of the Trichodesmium regulatory apparatus.

Replication of Nonautonomous Retroelements in Soybean Appears to be Both Recent and Common

USDA-ARS?s Scientific Manuscript database

Retrotransposons and their remnants often constitute more than 50% of higher plant genomes and have had major impacts on genome structure. Although extensively studied in monocot crops such as maize and rice, the impact of retrotransposons on major dicot crop genomes is not well documented. Here w...

Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon.

PubMed

Mitra, Rupak; Li, Xianghong; Kapusta, Aurélie; Mayhew, David; Mitra, Robi D; Feschotte, Cédric; Craig, Nancy L

2013-01-02

A revelation of the genomic age has been the contributions of the mobile DNA segments called transposable elements to chromosome structure, function, and evolution in virtually all organisms. Substantial fractions of vertebrate genomes derive from transposable elements, being dominated by retroelements that move via RNA intermediates. Although many of these elements have been inactivated by mutation, several active retroelements remain. Vertebrate genomes also contain substantial quantities and a high diversity of cut-and-paste DNA transposons, but no active representative of this class has been identified in mammals. Here we show that a cut-and-paste element called piggyBat, which has recently invaded the genome of the little brown bat (Myotis lucifugus) and is a member of the piggyBac superfamily, is active in its native form in transposition assays in bat and human cultured cells, as well as in the yeast Saccharomyces cerevisiae. Our study suggests that some DNA transposons are still actively shaping some mammalian genomes and reveals an unprecedented opportunity to study the mechanism, regulation, and genomic impact of cut-and-paste transposition in a natural mammalian host.

Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis.

PubMed

Kuwabara, Tomoko; Hsieh, Jenny; Muotri, Alysson; Yeo, Gene; Warashina, Masaki; Lie, Dieter Chichung; Moore, Lynne; Nakashima, Kinichi; Asashima, Makoto; Gage, Fred H

2009-09-01

In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded in the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.

Retrotransposons Mimic Germ Plasm Determinants to Promote Transgenerational Inheritance.

PubMed

Tiwari, Bhavana; Kurtz, Paula; Jones, Amanda E; Wylie, Annika; Amatruda, James F; Boggupalli, Devi Prasad; Gonsalvez, Graydon B; Abrams, John M

2017-10-09

Retrotransposons are a pervasive class of mobile elements present in the genomes of virtually all forms of life [1, 2]. In metazoans, these are preferentially active in the germline, which, in turn, mounts defenses that restrain their activity [3, 4]. Here we report that certain classes of retrotransposons ensure transgenerational inheritance by invading presumptive germ cells before they are formed. Using sensitized Drosophila and zebrafish models, we found that diverse classes of retrotransposons migrate to the germ plasm, a specialized region of the oocyte that prefigures germ cells and specifies the germline of descendants in the fertilized egg. In Drosophila, we found evidence for a "stowaway" model, whereby Tahre retroelements traffic to the germ plasm by mimicking oskar RNAs and engaging the Staufen-dependent active transport machinery. Consistent with this, germ plasm determinants attracted retroelement RNAs even when these components were ectopically positioned in bipolar oocytes. Likewise, vertebrate retrotransposons similarly migrated to the germ plasm in zebrafish oocytes. Together, these results suggest that germ plasm targeting represents a fitness strategy adopted by some retrotransposons to ensure transgenerational propagation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence for an uncommon alpha-actinin protein in Trichomonas vaginalis.

PubMed

Bricheux, G; Coffe, G; Pradel, N; Brugerolle, G

1998-09-15

As part of our ongoing project of identification of actin-binding proteins implicated in the cell transition (flagellate to amoeboid/adherent) of Trichomonas vaginalis, we have characterized an alpha-actinin-related protein in this parasite. The protein (P100) has a molecular mass of 100 kDa and an isoelectric point of 5.5. A monoclonal antibody raised against this protein co-localizes with the actin network. P100 gene transcripts are co-expressed with actin throughout the cell cycle. Analysis of the deduced protein sequence reveals three domains: an N-terminal actin-binding region; a central region rich in alpha-helix; and a C-terminal domain with Ca(2+)-binding capacity. Whereas the N- and C-terminal regions are well-conserved as compared to other alpha-actinins, we observe in the central region an atypical distribution of residues in five repeats. The sequence of the repeats does not show any homology with the rod domain of the other alpha-actinins, except for the first repeat which shows some similarity. The four other repeats of T. vaginalis P100 appear to result from a duplication event which is not detectable in the other sequences.

All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus.

PubMed Central

Klobutcher, L A; Swanton, M T; Donini, P; Prescott, D M

1981-01-01

In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of approximately 2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ... In Euplotes aediculatus, the sequences is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A-A-C-T-T-T-T-G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region: 5'-C4A4C4A4C4A4C4(X)17T-T-G-A-A ... 3'-G2T4G4T4G4T4G4T4G4T4G4(X)17A-A-C-T-T ... The length of the terminal repeat sequence for O. nova was confirmed in cloned macronuclear DNA molecules. Images PMID:6265931

Transposable element distribution, abundance and role in genome size variation in the genus Oryza.

PubMed

Zuccolo, Andrea; Sebastian, Aswathy; Talag, Jayson; Yu, Yeisoo; Kim, HyeRan; Collura, Kristi; Kudrna, Dave; Wing, Rod A

2007-08-29

The genus Oryza is composed of 10 distinct genome types, 6 diploid and 4 polyploid, and includes the world's most important food crop - rice (Oryza sativa [AA]). Genome size variation in the Oryza is more than 3-fold and ranges from 357 Mbp in Oryza glaberrima [AA] to 1283 Mbp in the polyploid Oryza ridleyi [HHJJ]. Because repetitive elements are known to play a significant role in genome size variation, we constructed random sheared small insert genomic libraries from 12 representative Oryza species and conducted a comprehensive study of the repetitive element composition, distribution and phylogeny in this genus. Particular attention was paid to the role played by the most important classes of transposable elements (Long Terminal Repeats Retrotransposons, Long interspersed Nuclear Elements, helitrons, DNA transposable elements) in shaping these genomes and in their contributing to genome size variation. We identified the elements primarily responsible for the most strikingly genome size variation in Oryza. We demonstrated how Long Terminal Repeat retrotransposons belonging to the same families have proliferated to very different extents in various species. We also showed that the pool of Long Terminal Repeat Retrotransposons is substantially conserved and ubiquitous throughout the Oryza and so its origin is ancient and its existence predates the speciation events that originated the genus. Finally we described the peculiar behavior of repeats in the species Oryza coarctata [HHKK] whose placement in the Oryza genus is controversial. Long Terminal Repeat retrotransposons are the major component of the Oryza genomes analyzed and, along with polyploidization, are the most important contributors to the genome size variation across the Oryza genus. Two families of Ty3-gypsy elements (RIRE2 and Atlantys) account for a significant portion of the genome size variations present in the Oryza genus.

A Novel Terminal-Repeat Retrotransposon in Miniature (TRIM) Is Massively Expressed in Echinococcus multilocularis Stem Cells

PubMed Central

Koziol, Uriel; Radio, Santiago; Smircich, Pablo; Zarowiecki, Magdalena; Fernández, Cecilia; Brehm, Klaus

2015-01-01

Taeniid cestodes (including the human parasites Echinococcus spp. and Taenia solium) have very few mobile genetic elements (MGEs) in their genome, despite lacking a canonical PIWI pathway. The MGEs of these parasites are virtually unexplored, and nothing is known about their expression and silencing. In this work, we report the discovery of a novel family of small nonautonomous long terminal repeat retrotransposons (also known as terminal-repeat retrotransposons in miniature, TRIMs) which we have named ta-TRIM (taeniid TRIM). ta-TRIMs are only the second family of TRIM elements discovered in animals, and are likely the result of convergent reductive evolution in different taxonomic groups. These elements originated at the base of the taeniid tree and have expanded during taeniid diversification, including after the divergence of closely related species such as Echinococcus multilocularis and Echinococcus granulosus. They are massively expressed in larval stages, from a small proportion of full-length copies and from isolated terminal repeats that show transcriptional read-through into downstream regions, generating novel noncoding RNAs and transcriptional fusions to coding genes. In E. multilocularis, ta-TRIMs are specifically expressed in the germinative cells (the somatic stem cells) during asexual reproduction of metacestode larvae. This would provide a developmental mechanism for insertion of ta-TRIMs into cells that will eventually generate the adult germ line. Future studies of active and inactive ta-TRIM elements could give the first clues on MGE silencing mechanisms in cestodes. PMID:26133390

The crystal structure of a partial mouse Notch-1 ankyrin domain: Repeats 4 through 7 preserve an ankyrin fold

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

Lubman, Olga Y.; Kopan, Raphael; Waksman, Gabriel

Folding and stability of proteins containing ankyrin repeats (ARs) is of great interest because they mediate numerous protein-protein interactions involved in a wide range of regulatory cellular processes. Notch, an ankyrin domain containing protein, signals by converting a transcriptional repression complex into an activation complex. The Notch ANK domain is essential for Notch function and contains seven ARs. Here, we present the 2.2 {angstrom} crystal structure of ARs 4-7 from mouse Notch 1 (m1ANK). These C-terminal repeats were resistant to degradation during crystallization, and their secondary and tertiary structures are maintained in the absence of repeats 1-3. The crystallized fragmentmore » adopts a typical ankyrin fold including the poorly conserved seventh AR, as seen in the Drosophila Notch ANK domain (dANK). The structural preservation and stability of the C-terminal repeats shed a new light onto the mechanism of hetero-oligomeric assembly during Notch-mediated transcriptional activation.« less

Differential Accumulation of Retroelements and Diversification of NB-LRR Disease Resistance Genes in Duplicated Regions Following Polyploidy in the Ancestor of Soybean

USDA-ARS?s Scientific Manuscript database

The genomes of most flowering plants have undergone polyploidization at some point in their evolution. How such polyploidization events have impacted the subsequent evolution of genome structure is poorly understood. We sequenced two homoeologous regions in soybean (Glycine max), which underwent a...

Analysis of BAC end sequences in oak, a keystone forest tree species, providing insight into the composition of its genome

PubMed Central

2011-01-01

Background One of the key goals of oak genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of forests to increase their health and productivity. Deep-coverage large-insert genomic libraries are a crucial tool for attaining this objective. We report herein the construction of a BAC library for Quercus robur, its characterization and an analysis of BAC end sequences. Results The EcoRI library generated consisted of 92,160 clones, 7% of which had no insert. Levels of chloroplast and mitochondrial contamination were below 3% and 1%, respectively. Mean clone insert size was estimated at 135 kb. The library represents 12 haploid genome equivalents and, the likelihood of finding a particular oak sequence of interest is greater than 99%. Genome coverage was confirmed by PCR screening of the library with 60 unique genetic loci sampled from the genetic linkage map. In total, about 20,000 high-quality BAC end sequences (BESs) were generated by sequencing 15,000 clones. Roughly 5.88% of the combined BAC end sequence length corresponded to known retroelements while ab initio repeat detection methods identified 41 additional repeats. Collectively, characterized and novel repeats account for roughly 8.94% of the genome. Further analysis of the BESs revealed 1,823 putative genes suggesting at least 29,340 genes in the oak genome. BESs were aligned with the genome sequences of Arabidopsis thaliana, Vitis vinifera and Populus trichocarpa. One putative collinear microsyntenic region encoding an alcohol acyl transferase protein was observed between oak and chromosome 2 of V. vinifera. Conclusions This BAC library provides a new resource for genomic studies, including SSR marker development, physical mapping, comparative genomics and genome sequencing. BES analysis provided insight into the structure of the oak genome. These sequences will be used in the assembly of a future genome sequence for oak. PMID:21645357

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin

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

Hill, Shannon E.; Nguyen, Elaine; Donegan, Rebecca K.

2017-11-01

Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains.more » Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.« less

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin.

PubMed

Hill, Shannon E; Nguyen, Elaine; Donegan, Rebecca K; Patterson-Orazem, Athéna C; Hazel, Anthony; Gumbart, James C; Lieberman, Raquel L

2017-11-07

Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Novel Terminal-Repeat Retrotransposon in Miniature (TRIM) Is Massively Expressed in Echinococcus multilocularis Stem Cells.

PubMed

Koziol, Uriel; Radio, Santiago; Smircich, Pablo; Zarowiecki, Magdalena; Fernández, Cecilia; Brehm, Klaus

2015-07-01

Taeniid cestodes (including the human parasites Echinococcus spp. and Taenia solium) have very few mobile genetic elements (MGEs) in their genome, despite lacking a canonical PIWI pathway. The MGEs of these parasites are virtually unexplored, and nothing is known about their expression and silencing. In this work, we report the discovery of a novel family of small nonautonomous long terminal repeat retrotransposons (also known as terminal-repeat retrotransposons in miniature, TRIMs) which we have named ta-TRIM (taeniid TRIM). ta-TRIMs are only the second family of TRIM elements discovered in animals, and are likely the result of convergent reductive evolution in different taxonomic groups. These elements originated at the base of the taeniid tree and have expanded during taeniid diversification, including after the divergence of closely related species such as Echinococcus multilocularis and Echinococcus granulosus. They are massively expressed in larval stages, from a small proportion of full-length copies and from isolated terminal repeats that show transcriptional read-through into downstream regions, generating novel noncoding RNAs and transcriptional fusions to coding genes. In E. multilocularis, ta-TRIMs are specifically expressed in the germinative cells (the somatic stem cells) during asexual reproduction of metacestode larvae. This would provide a developmental mechanism for insertion of ta-TRIMs into cells that will eventually generate the adult germ line. Future studies of active and inactive ta-TRIM elements could give the first clues on MGE silencing mechanisms in cestodes. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

PubMed Central

Pang, Xiuhua; Aigle, Bertrand; Girardet, Jean-Michel; Mangenot, Sophie; Pernodet, Jean-Luc; Decaris, Bernard; Leblond, Pierre

2004-01-01

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the β-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production. PMID:14742212

An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

PubMed

Ishikawa, Yoshihiro; Bächinger, Hans Peter

2013-11-01

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

Interstitial telomeric sequences in vertebrate chromosomes: Origin, function, instability and evolution.

PubMed

Bolzán, Alejandro D

2017-07-01

By definition, telomeric sequences are located at the very ends or terminal regions of chromosomes. However, several vertebrate species show blocks of (TTAGGG)n repeats present in non-terminal regions of chromosomes, the so-called interstitial telomeric sequences (ITSs), interstitial telomeric repeats or interstitial telomeric bands, which include those intrachromosomal telomeric-like repeats located near (pericentromeric ITSs) or within the centromere (centromeric ITSs) and those telomeric repeats located between the centromere and the telomere (i.e., truly interstitial telomeric sequences) of eukaryotic chromosomes. According with their sequence organization, localization and flanking sequences, ITSs can be classified into four types: 1) short ITSs, 2) subtelomeric ITSs, 3) fusion ITSs, and 4) heterochromatic ITSs. The first three types have been described mainly in the human genome, whereas heterochromatic ITSs have been found in several vertebrate species but not in humans. Several lines of evidence suggest that ITSs play a significant role in genome instability and evolution. This review aims to summarize our current knowledge about the origin, function, instability and evolution of these telomeric-like repeats in vertebrate chromosomes. Copyright © 2017 Elsevier B.V. All rights reserved.

An ancient trans-kingdom horizontal transfer of Penelope -like retroelements from arthropods to conifers

Treesearch

Xuan Lin; Nurul Faridi; Claudio Casola

2016-01-01

Comparative genomics analyses empowered by the wealth of sequenced genomes have revealed numerous instances of horizontal DNA transfers between distantly related species. In  eukaryotes, repetitive DNA sequences known as transposable elements (TEs) are especially prone to  move across species boundaries. Such horizontal transposon transfers, or HTTs, are relatively  ...

A Novel MAPT Mutation, G55R, in a Frontotemporal Dementia Patient Leads to Altered Tau Function

PubMed Central

Guzman, Elmer; Barczak, Anna; Chodakowska-Żebrowska, Małgorzata; Barcikowska, Maria; Feinstein, Stuart

2013-01-01

Over two dozen mutations in the gene encoding the microtubule associated protein tau cause a variety of neurodegenerative dementias known as tauopathies, including frontotemporal dementia (FTD), PSP, CBD and Pick's disease. The vast majority of these mutations map to the C-terminal region of tau possessing microtubule assembly and microtubule dynamics regulatory activities as well as the ability to promote pathological tau aggregation. Here, we describe a novel and non-conservative tau mutation (G55R) mapping to an alternatively spliced exon encoding part of the N-terminal region of the protein in a patient with the behavioral variant of FTD. Although less well understood than the C-terminal region of tau, the N-terminal region can influence both MT mediated effects as well as tau aggregation. The mutation changes an uncharged glycine to a basic arginine in the midst of a highly conserved and very acidic region. In vitro, 4-repeat G55R tau nucleates microtubule assembly more effectively than wild-type 4-repeat tau; surprisingly, this effect is tau isoform specific and is not observed in a 3-repeat G55R tau versus 3-repeat wild-type tau comparison. In contrast, the G55R mutation has no effect upon the abilities of tau to regulate MT growing and shortening dynamics or to aggregate. Additionally, the mutation has no effect upon kinesin translocation in a microtubule gliding assay. Together, (i) we have identified a novel tau mutation mapping to a mutation deficient region of the protein in a bvFTD patient, and (ii) the G55R mutation affects the ability of tau to nucleate microtubule assembly in vitro in a 4-repeat tau isoform specific manner. This altered capability could markedly affect in vivo microtubule function and neuronal cell biology. We consider G55R to be a candidate mutation for bvFTD since additional criteria required to establish causality are not yet available for assessment. PMID:24086739

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

PubMed

Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

2018-04-27

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

Variation, Repetition, and Choice

ERIC Educational Resources Information Center

Abreu-Rodrigues, Josele; Lattal, Kennon A.; dos Santos, Cristiano V.; Matos, Ricardo A.

2005-01-01

Experiment 1 investigated the controlling properties of variability contingencies on choice between repeated and variable responding. Pigeons were exposed to concurrent-chains schedules with two alternatives. In the REPEAT alternative, reinforcers in the terminal link depended on a single sequence of four responses. In the VARY alternative, a…

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

Follis, Kathryn E.; York, Joanne; Nunberg, Jack H.

The fusion subunit of the SARS-CoV S glycoprotein contains two regions of hydrophobic heptad-repeat amino acid sequences that have been shown in biophysical studies to form a six-helix bundle structure typical of the fusion-active core found in Class I viral fusion proteins. Here, we have applied serine-scanning mutagenesis to the C-terminal-most heptad-repeat region in the SARS-CoV S glycoprotein to investigate the functional role of this region in membrane fusion. We show that hydrophobic sidechains at a and d positions only within the short helical segment of the C-terminal heptad-repeat region (I1161, I1165, L1168, A1172, and L1175) are critical for cell-cellmore » fusion. Serine mutations at outlying heptad-repeat residues that form an extended chain in the core structure (V1158, L1179, and L1182) do not affect fusogenicity. Our study provides genetic evidence for the important role of {alpha}-helical packing in promoting S glycoprotein-mediated membrane fusion.« less

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats.

PubMed

Hasegawa, Morio; Toma-Fukai, Sachiko; Kim, Jun-Dal; Fukamizu, Akiyoshi; Shimizu, Toshiyuki

2014-05-21

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-L-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-L-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Tyrosine Sulfate Domain of Fibromodulin Binds Collagen and Enhances Fibril Formation.

PubMed

Tillgren, Viveka; Mörgelin, Matthias; Önnerfjord, Patrik; Kalamajski, Sebastian; Aspberg, Anders

2016-11-04

Small leucine-rich proteoglycans interact with other extracellular matrix proteins and are important regulators of matrix assembly. Fibromodulin has a key role in connective tissues, binding collagen through two identified binding sites in its leucine-rich repeat domain and regulating collagen fibril formation in vitro and in vivo Some nine tyrosine residues in the fibromodulin N-terminal domain are O-sulfated, a posttranslational modification often involved in protein interactions. The N-terminal domain mimics heparin, binding proteins with clustered basic amino acid residues. Because heparin affects collagen fibril formation, we investigated whether tyrosine sulfate is involved in fibromodulin interactions with collagen. Using full-length fibromodulin and its N-terminal tyrosine-sulfated domain purified from tissue, as well as recombinant fibromodulin fragments, we found that the N-terminal domain binds collagen. The tyrosine-sulfated domain and the leucine-rich repeat domain both bound to three specific sites along the collagen type I molecule, at the N terminus and at 100 and 220 nm from the N terminus. The N-terminal domain shortened the collagen fibril formation lag phase and tyrosine sulfation was required for this effect. The isolated leucine-rich repeat domain inhibited the fibril formation rate, and full-length fibromodulin showed a combination of these effects. The fibrils formed in the presence of fibromodulin or its fragments showed more organized structure. Fibromodulin and its tyrosine sulfate domain remained bound on the formed fiber. Taken together, this suggests a novel, regulatory function for tyrosine sulfation in collagen interaction and control of fibril formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of Spindle Checkpoint Kinase Mps1 Reveals Domain with Functional and Structural Similarities to Tetratricopeptide Repeat Motifs of Bub1 and BubR1 Checkpoint Kinases*

PubMed Central

Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L.; Landry, Christian R.; Bolanos-Garcia, Victor M.; Elowe, Sabine

2012-01-01

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region. PMID:22187426

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

PubMed

Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L; Landry, Christian R; Bolanos-Garcia, Victor M; Elowe, Sabine

2012-02-17

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

Long interspersed nuclear element-1 retroelements are expressed in patients with systemic autoimmune disease and induce type I interferon

PubMed Central

Mavragani, Clio P.; Sagalovskiy, Irina; Guo, Qiu; Nezos, Adrianos; Kapsogeorgou, Efstathia K.; Lu, Pin; Zhou, Jun Liang; Kirou, Kyriakos A.; Seshan, Surya V.; Moutsopoulos, Haralampos M.; Crow, Mary K.

2016-01-01

Objective Increased type I interferon (IFN-I) and a broad signature of IFN-I-induced gene transcripts are observed in patients with SLE and other systemic autoimmune diseases. To identify disease-relevant triggers of the IFN-I pathway we investigated whether endogenous virus-like genomic repeat elements, normally silent, might be expressed in patients with systemic autoimmune disease, activate an innate immune response and induce IFN-I. Methods Expression of IFN-I and long interspersed nuclear element-1 (LINE-1; L1) was studied in kidney tissue from lupus patients and minor salivary gland (MSG) tissue from patients with primary Sjogren’s syndrome (SS) by PCR, western blot and immunohistochemistry. Induction of IFN-I by L1 was investigated by transfection of plasmacytoid dendritic cells (pDCs) or monocytes with an L1-encoding plasmid or L1 RNA. Involvement of innate immune pathways and altered L1 methylation were assessed. Results L1 mRNA transcripts were increased in lupus nephritis kidneys and in MSG from SS patients and correlated with IFN-I expression and L1 DNA demethylation. L1 open reading frame 1/p40 protein and IFNβ were expressed in MSG ductal epithelial cells and in lupus kidneys, and IFNα was detected in infiltrating pDCs. Transfection of pDCs or monocytes with L1-encoding DNA or RNA induced IFN-I. Inhibition of TLR7/8 reduced L1 induction of IFNα in pDCs and an inhibitor of IKKε/TBK1 abrogated induction of IFN-I by L1 RNA in monocytes. Conclusion L1 genomic repeat elements represent endogenous nucleic acid triggers of the IFN-I pathway in SLE and SS and may contribute to initiation or amplification of autoimmune disease. PMID:27338297

24 CFR 880.607 - Termination of tenancy and modification of lease.

Code of Federal Regulations, 2011 CFR

2011-04-01

... CONSTRUCTION Management § 880.607 Termination of tenancy and modification of lease. (a) Applicability. The... lease; or (B) Repeated minor violations of the lease that disrupt the livability of the building... leased premises and related facilities; interfere with the management of the building or have an adverse...

Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

PubMed

Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

2012-07-01

Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

Bioinformatic flowchart and database to investigate the origins and diversity of Clan AA peptidases

PubMed Central

Llorens, Carlos; Futami, Ricardo; Renaud, Gabriel; Moya, Andrés

2009-01-01

Background Clan AA of aspartic peptidases relates the family of pepsin monomers evolutionarily with all dimeric peptidases encoded by eukaryotic LTR retroelements. Recent findings describing various pools of single-domain nonviral host peptidases, in prokaryotes and eukaryotes, indicate that the diversity of clan AA is larger than previously thought. The ensuing approach to investigate this enzyme group is by studying its phylogeny. However, clan AA is a difficult case to study due to the low similarity and different rates of evolution. This work is an ongoing attempt to investigate the different clan AA families to understand the cause of their diversity. Results In this paper, we describe in-progress database and bioinformatic flowchart designed to characterize the clan AA protein domain based on all possible protein families through ancestral reconstructions, sequence logos, and hidden markov models (HMMs). The flowchart includes the characterization of a major consensus sequence based on 6 amino acid patterns with correspondence with Andreeva's model, the structural template describing the clan AA peptidase fold. The set of tools is work in progress we have organized in a database within the GyDB project, referred to as Clan AA Reference Database . Conclusion The pre-existing classification combined with the evolutionary history of LTR retroelements permits a consistent taxonomical collection of sequence logos and HMMs. This set is useful for gene annotation but also a reference to evaluate the diversity of, and the relationships among, the different families. Comparisons among HMMs suggest a common ancestor for all dimeric clan AA peptidases that is halfway between single-domain nonviral peptidases and those coded by Ty3/Gypsy LTR retroelements. Sequence logos reveal how all clan AA families follow similar protein domain architecture related to the peptidase fold. In particular, each family nucleates a particular consensus motif in the sequence position related to the flap. The different motifs constitute a network where an alanine-asparagine-like variable motif predominates, instead of the canonical flap of the HIV-1 peptidase and closer relatives. Reviewers This article was reviewed by Daniel H. Haft, Vladimir Kapitonov (nominated by Jerry Jurka), and Ben M. Dunn (nominated by Claus Wilke). PMID:19173708

Comparative Characterization of Complete and Truncated Forms of Lactobacillus amylovorus α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding

PubMed Central

Rodriguez Sanoja, R.; Morlon-Guyot, J.; Jore, J.; Pintado, J.; Juge, N.; Guyot, J. P.

2000-01-01

Two constructs derived from the α-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyAΔ) forms of α-amylase; AmyAΔ lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyAΔ exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and α-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the KM and Vmax values of AmyAΔ were slightly higher than those of AmyA, whereas the thermal stability of AmyAΔ was lower than that of AmyA. In contrast to AmyA, AmyAΔ is unable to bind to β-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyAΔ cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity. PMID:10919790

Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes

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

Ono, M.

1986-06-01

By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identifiedmore » as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.« less

The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis

PubMed Central

Silva, Éverton F.; Medeiros, Marco A.; McBride, Alan J. A.; Matsunaga, Jim; Esteves, Gabriela S.; Ramos, João G. R.; Santos, Cleiton S.; Croda, Júlio; Homma, Akira; Dellagostin, Odir A.; Haake, David A.; Reis, Mitermayer G.; Ko, Albert I.

2007-01-01

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund’s adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P <0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis. PMID:17629368

The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis.

PubMed

Silva, Everton F; Medeiros, Marco A; McBride, Alan J A; Matsunaga, Jim; Esteves, Gabriela S; Ramos, João G R; Santos, Cleiton S; Croda, Júlio; Homma, Akira; Dellagostin, Odir A; Haake, David A; Reis, Mitermayer G; Ko, Albert I

2007-08-14

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund's adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P<0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.

Identification of Novel Inverted Terminal Repeat (ITR) Deletions of Human Adenovirus (AD) From Infected Host: Virulent Ads Containing Mixed Populations of Genomic Sequences

DTIC Science & Technology

2006-11-01

terminal repetition of adenvirus type 4 DNA. Gene 18:329-334. 20. Van der Veen , J., and J. H. Dijkman . 1962. Association of type 21 adenovirus with acute respiratory illness in military recruits. Am J Hyg 76:149-159.

Engineering the N-terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose

DOE PAGES

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; ...

2016-12-26

Here, CelA is the most abundant enzyme secreted by Caldicellulosiruptor bescii and has been shown to outperform mixtures of commercially available exo- and endoglucanases in vitro. CelA contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. Here, repeated aspartate residues were introduced into the N-terminal ends of CelA GH9 and GH48 domains to improve secretion efficiency and/or catalytic efficiency of CelA. Among several constructs, the highest activity on carboxymethylcellulose (CMC), 0.81 ± 0.03 mg/mL was observed for the C.more » bescii strain containing CelA with 5-aspartate tag at the N-terminal end of GH9 domain – an 82% increase over wild type CelA. In addition, Expression of CelA with N-terminal repeated aspartate residues in C. bescii results in a dramatic increase in its ability to grow on Avicel.« less

The N-terminal Ankyrin Repeat Domain Is Not Required for Electrophile and Heat Activation of the Purified Mosquito TRPA1 Receptor*

PubMed Central

2016-01-01

Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing; some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional because they responded to the electrophilic compounds allyl isothiocyanate and cinnamaldehyde as well as heat. The proteins' similar intrinsic fluorescence properties and corresponding quenching when activated by allyl isothiocyanate or heat suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that AgTRPA1 is an inherent thermo- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog, the N-terminal domain may tune the response but is not required for the activation by these stimuli. PMID:27875296

Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus.

PubMed Central

Maurer, B; Bannert, H; Darai, G; Flügel, R M

1988-01-01

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae. Images PMID:2451755

Evaluation of genetic and metabolic role of SKIP11 in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Hassan, Muhammad Naeem ul; Ismail, Ismanizan

2015-09-01

Most of the regulatory proteins are degraded by 26S proteasome complex, only when they are tagged by Ubiquitin. A complex of four proteins, SKP1-Cullin-Ring box-F box (SCF) catalyses the final step to link the Ubiquitin tag with the target proteins. SCF complex interacts with the target proteins through F-box proteins, which confer the overall substrate specificity to the complex. F-box proteins, one of the largest family of proteins in plants have an N-terminal F-box domain and variable C-terminal domains, like leucine-rich repeat, WD-40 repeat and the kelch-repeat domains. In this study, we analysed the role of SKIP11, a kelch containing F-box protein (KFB) from Arabidopsis thaliana, by using reverse genetics strategy. The results show that SKIP11 is involved in the down-regulation of oxylipin pathway, possibly through the degradation of enzymes or/ and the regulatory factors of the pathway.

The 8th and 9th tandem spectrin-like repeats of utrophin cooperatively form a functional unit to interact with polarity-regulating kinase PAR-1b.

PubMed

Yamashita, Kazunari; Suzuki, Atsushi; Satoh, Yoshinori; Ide, Mariko; Amano, Yoshiko; Masuda-Hirata, Maki; Hayashi, Yukiko K; Hamada, Keisuke; Ogata, Kazuhiro; Ohno, Shigeo

2010-01-01

Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin-DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain. Copyright 2009 Elsevier Inc. All rights reserved.

Oncogenic LINE 1 Retroelements Sustain Prostate Tumor Cells and Promote Metastatic Progression

DTIC Science & Technology

2016-12-01

Tillotson University in Austin , Texas and provides a summer research experience for them where they learn about prostate cancer research , as well as...PRINCIPAL INVESTIGATOR: Denise S. O’Keefe, Ph.D. CONTRACTING ORGANIZATION: UNIVERSITY OF TEXAS , San Antonio SAN ANTONIO TX 78229-3901 REPORT DATE...December 2016 TYPE OF REPORT: Final PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012

Oncogenic LINE-1 Retroelements Sustain Prostate Tumor Cells and Promote Metastatic Progression

DTIC Science & Technology

2015-10-01

elements in prostate cancer contribute to its progression by activating oncogenic DNA sequences, or silencing tumor suppressor like sequences. We have...prostate cancer cells. Experiments are ongoing to determine if PIWIL-1 expression in prostate cancer cells will reduce their growth, thereby providing...proof of principle for future gene-based therapeutics for this cancer . 15. SUBJECT TERMS Prostate cancer , LINE-1, PIWIL-1, retrotransposons 16

Analysis of genetic diversity in pigeon pea germplasm using retrotransposon-based molecular markers.

PubMed

Maneesha; Upadhyaya, Kailash C

2017-09-01

Pigeon pea (Cajanus cajan), an important legume crop is predominantly cultivated in tropical and subtropical regions of Asia and Africa. It is normally considered to have a low degree of genetic diversity, an impediment in undertaking crop improvement programmes.We have analysed genetic polymorphism of domesticated pigeon pea germplasm (47 accessions) across the world using earlier characterized panzee retrotransposon-based molecularmarkers. Itwas conjectured that since retrotransposons are interspersed throughout the genome, retroelements-based markers would be able to uncover polymorphism possibly inherent in the diversity of retroelement sequences. Two PCR-based techniques, sequence-specific amplified polymorphism (SSAP) and retrotransposon microsatellite amplified polymorphism (REMAP) were utilized for the analyses.We show that a considerable degree of polymorphism could be detected using these techniques. Three primer combinations in SSAP generated 297 amplified products across 47 accessions with an average of 99 amplicons per assay. Degree of polymorphism varied from 84-95%. In the REMAP assays, the number of amplicons was much less but up to 73% polymorphism could be detected. On the basis of similarity coefficients, dendrograms were constructed. The results demonstrate that the retrotransposon-based markers could serve as a better alternative for the assessment of genetic diversity in crops with apparent low genetic base.

All y'all need to know 'bout retroelements in cancer.

PubMed

Belancio, Victoria P; Roy-Engel, Astrid M; Deininger, Prescott L

2010-08-01

Genetic instability is one of the principal hallmarks and causative factors in cancer. Human transposable elements (TE) have been reported to cause human diseases, including several types of cancer through insertional mutagenesis of genes critical for preventing or driving malignant transformation. In addition to retrotransposition-associated mutagenesis, TEs have been found to contribute even more genomic rearrangements through non-allelic homologous recombination. TEs also have the potential to generate a wide range of mutations derivation of which is difficult to directly trace to mobile elements, including double strand breaks that may trigger mutagenic genomic rearrangements. Genome-wide hypomethylation of TE promoters and significantly elevated TE expression in almost all human cancers often accompanied by the loss of critical DNA sensing and repair pathways suggests that the negative impact of mobile elements on genome stability should increase as human tumors evolve. The biological consequences of elevated retroelement expression, such as the rate of their amplification, in human cancers remain obscure, particularly, how this increase translates into disease-relevant mutations. This review is focused on the cellular mechanisms that control human TE-associated mutagenesis in cancer and summarizes the current understanding of TE contribution to genetic instability in human malignancies. Copyright © 2010 Elsevier Ltd. All rights reserved.

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

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

Le Coq, Johanne; Ghosh, Partho

2012-06-19

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein,more » TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd ({approx}16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10{sup 20} potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.« less

Envelope-like retrotransposons in the plant kingdom: evidence of their presence in gymnosperms (Pinus pinaster).

PubMed

Miguel, Célia; Simões, Marta; Oliveira, Maria Margarida; Rocheta, Margarida

2008-11-01

Retroviruses differ from retrotransposons due to their infective capacity, which depends critically on the encoded envelope. Some plant retroelements contain domains reminiscent of the env of animal retroviruses but the number of such elements described to date is restricted to angiosperms. We show here the first evidence of the presence of putative env-like gene sequences in a gymnosperm species, Pinus pinaster (maritime pine). Using a degenerate primer approach for conserved domains of RNaseH gene, three clones from putative envelope-like retrotransposons (PpRT2, PpRT3, and PpRT4) were identified. The env-like sequences of P. pinaster clones are predicted to encode proteins with transmembrane domains. These sequences showed identity scores of up to 30% with env-like sequences belonging to different organisms. A phylogenetic analysis based on protein alignment of deduced aminoacid sequences revealed that these clones clustered with env-containing plant retrotransposons, as well as with retrotransposons from invertebrate organisms. The differences found among the sequences of maritime pine clones isolated here suggest the existence of different putative classes of env-like retroelements. The identification for the first time of env-like genes in a gymnosperm species may support the ancestrality of retroviruses among plants shedding light on their role in plant evolution.

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

PubMed Central

Le Coq, Johanne; Ghosh, Partho

2011-01-01

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 1020 potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation. PMID:21873231

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement.

PubMed

Le Coq, Johanne; Ghosh, Partho

2011-08-30

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10(20) potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.

E622, a miniature, virulence-associated mobile element.

PubMed

Stavrinides, John; Kirzinger, Morgan W B; Beasley, Federico C; Guttman, David S

2012-01-01

Miniature inverted terminal repeat elements (MITEs) are nonautonomous mobile elements that have a significant impact on bacterial evolution. Here we characterize E622, a 611-bp virulence-associated MITE from Pseudomonas syringae, which contains no coding region but has almost perfect 168-bp inverted repeats. Using an antibiotic coupling assay, we show that E622 is transposable and can mobilize an antibiotic resistance gene contained between its borders. Its predicted parent element, designated TnE622, has a typical transposon structure with a three-gene operon, consisting of resolvase, integrase, and exeA-like genes, which is bounded by the same terminal inverted repeats as E622. A broader genome level survey of the E622/TnE622 inverted repeats identified homologs in Pseudomonas, Salmonella, Shewanella, Erwinia, Pantoea, and the cyanobacteria Nostoc and Cyanothece, many of which appear to encompass known virulence genes, including genes encoding toxins, enzymes, and type III secreted effectors. Its association with niche-specific genetic determinants, along with its persistence and evolutionary diversification, indicates that this mobile element family has played a prominent role in the evolution of many agriculturally and clinically relevant pathogenic bacteria.

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species

PubMed Central

Wu, Chengcang; Proestou, Dina; Carter, Dorothy; Nicholson, Erica; Santos, Filippe; Zhao, Shaying; Zhang, Hong-Bin; Goldsmith, Marian R

2009-01-01

Background Manduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date. Results We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences. Conclusion The high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes. PMID:19558662

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein*S⃞

PubMed Central

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L.; Levin, Henry L.

2008-01-01

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the γ-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining. PMID:18397885

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.

PubMed

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L; Levin, Henry L

2008-06-06

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

Evolution of genes and repeats in the Nimrod superfamily.

PubMed

Somogyi, Kálmán; Sipos, Botond; Pénzes, Zsolt; Kurucz, Eva; Zsámboki, János; Hultmark, Dan; Andó, István

2008-11-01

The recently identified Nimrod superfamily is characterized by the presence of a special type of EGF repeat, the NIM repeat, located right after a typical CCXGY/W amino acid motif. On the basis of structural features, nimrod genes can be divided into three types. The proteins encoded by Draper-type genes have an EMI domain at the N-terminal part and only one copy of the NIM motif, followed by a variable number of EGF-like repeats. The products of Nimrod B-type and Nimrod C-type genes (including the eater gene) have different kinds of N-terminal domains, and lack EGF-like repeats but contain a variable number of NIM repeats. Draper and Nimrod C-type (but not Nimrod B-type) proteins carry a transmembrane domain. Several members of the superfamily were claimed to function as receptors in phagocytosis and/or binding of bacteria, which indicates an important role in the cellular immunity and the elimination of apoptotic cells. In this paper, the evolution of the Nimrod superfamily is studied with various methods on the level of genes and repeats. A hypothesis is presented in which the NIM repeat, along with the EMI domain, emerged by structural reorganizations at the end of an EGF-like repeat chain, suggesting a mechanism for the formation of novel types of repeats. The analyses revealed diverse evolutionary patterns in the sequences containing multiple NIM repeats. Although in the Nimrod B and Nimrod C proteins show characteristics of independent evolution, many internal NIM repeats in Eater sequences seem to have undergone concerted evolution. An analysis of the nimrod genes has been performed using phylogenetic and other methods and an evolutionary scenario of the origin and diversification of the Nimrod superfamily is proposed. Our study presents an intriguing example how the evolution of multigene families may contribute to the complexity of the innate immune response.

DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica

PubMed Central

Wang, Rui; Li, Ming; Gong, Luyao; Hu, Songnian; Xiang, Hua

2016-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) acquire new spacers to generate adaptive immunity in prokaryotes. During spacer integration, the leader-preceded repeat is always accurately duplicated, leading to speculations of a repeat-length ruler. Here in Haloarcula hispanica, we demonstrate that the accurate duplication of its 30-bp repeat requires two conserved mid-repeat motifs, AACCC and GTGGG. The AACCC motif was essential and needed to be ∼10 bp downstream from the leader-repeat junction site, where duplication consistently started. Interestingly, repeat duplication terminated sequence-independently and usually with a specific distance from the GTGGG motif, which seemingly served as an anchor site for a molecular ruler. Accordingly, altering the spacing between the two motifs led to an aberrant duplication size (29, 31, 32 or 33 bp). We propose the adaptation complex may recognize these mid-repeat elements to enable measuring the repeat DNA for spacer integration. PMID:27085805

Glider and Vision: two new families of miniature inverted-repeat transposable elements in Xenopus laevis genome.

PubMed

Lepetit, D; Pasquet, S; Olive, M; Thézé, N; Thiébaud, P

2000-01-01

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the alpha-tropomyosin (alpha-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated alpha-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the Xenopus laevis genome. Every MITEs elements but two described in our study are found either in 5' or in 3' regulatory regions of genes suggesting a potential role in gene regulation.

Quantitative analysis of TALE-DNA interactions suggests polarity effects.

PubMed

Meckler, Joshua F; Bhakta, Mital S; Kim, Moon-Soo; Ovadia, Robert; Habrian, Chris H; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David J; Baldwin, Enoch P

2013-04-01

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN > NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

Chlorovirus Skp1-binding ankyrin repeat protein interplay and mimicry of cellular ubiquitin ligase machinery.

PubMed

Noel, Eric A; Kang, Ming; Adamec, Jiri; Van Etten, James L; Oyler, George A

2014-12-01

The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

26 CFR 1.507-2 - Special rules; transfer to, or operation as, public charity.

Code of Federal Regulations, 2010 CFR

2010-04-01

... the seeking of advice by the transferee from, or the giving of advice by, any donor after the assets... willful repeated acts (or failures to act) or a willful and flagrant act (or failure to act) giving rise... apply to such a termination, a private foundation which makes such a termination is not required to give...

26 CFR 1.507-2 - Special rules; transfer to, or operation as, public charity.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the seeking of advice by the transferee from, or the giving of advice by, any donor after the assets... willful repeated acts (or failures to act) or a willful and flagrant act (or failure to act) giving rise... apply to such a termination, a private foundation which makes such a termination is not required to give...

Structure and Functional Characterization of the RNA-Binding Element of the NLRX1 Innate Immune Modulator

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

Hong, Minsun; Yoon, Sung-il; Wilson, Ian A.

2012-06-20

Mitochondrial NLRX1 is a member of the family of nucleotide-binding domain and leucine-rich-repeat-containing proteins (NLRs) that mediate host innate immunity as intracellular surveillance sensors against common molecular patterns of invading pathogens. NLRX1 functions in antiviral immunity, but the molecular mechanism of its ligand-induced activation is largely unknown. The crystal structure of the C-terminal fragment (residues 629975) of human NLRX1 (cNLRX1) at 2.65 {angstrom} resolution reveals that cNLRX1 consists of an N-terminal helical (LRRNT) domain, central leucine-rich repeat modules (LRRM), and a C-terminal three-helix bundle (LRRCT). cNLRX1 assembles into a compact hexameric architecture that is stabilized by intersubunit and interdomain interactionsmore » of LRRNT and LRRCT in the trimer and dimer components of the hexamer, respectively. Furthermore, we find that cNLRX1 interacts directly with RNA and supports a role for NLRX1 in recognition of intracellular viral RNA in antiviral immunity.« less

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

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.

Here, CelA is the most abundant enzyme secreted by Caldicellulosiruptor bescii and has been shown to outperform mixtures of commercially available exo- and endoglucanases in vitro. CelA contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. Here, repeated aspartate residues were introduced into the N-terminal ends of CelA GH9 and GH48 domains to improve secretion efficiency and/or catalytic efficiency of CelA. Among several constructs, the highest activity on carboxymethylcellulose (CMC), 0.81 ± 0.03 mg/mL was observed for the C.more » bescii strain containing CelA with 5-aspartate tag at the N-terminal end of GH9 domain – an 82% increase over wild type CelA. In addition, Expression of CelA with N-terminal repeated aspartate residues in C. bescii results in a dramatic increase in its ability to grow on Avicel.« less

ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.

PubMed

You, Zhongsheng; Chahwan, Charly; Bailis, Julie; Hunter, Tony; Russell, Paul

2005-07-01

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.

The self primer of the long terminal repeat retrotransposon Tf1 is not removed during reverse transcription.

PubMed

Atwood-Moore, Angela; Yan, Kenneth; Judson, Robert L; Levin, Henry L

2006-08-01

The long terminal repeat retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5' end of its own transcript. We tested whether the self primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self primer. This suggests that integration of the cDNA relies on mechanisms other than reverse transcription to remove the primer.

The mouse genome displays highly dynamic populations of KRAB-zinc finger protein genes and related genetic units

PubMed Central

Kauzlaric, Annamaria; Ecco, Gabriela; Cassano, Marco; Duc, Julien; Imbeault, Michael; Trono, Didier

2017-01-01

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression. PMID:28334004

The mouse genome displays highly dynamic populations of KRAB-zinc finger protein genes and related genetic units.

PubMed

Kauzlaric, Annamaria; Ecco, Gabriela; Cassano, Marco; Duc, Julien; Imbeault, Michael; Trono, Didier

2017-01-01

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression.

Human seizures self-terminate across spatial scales via a critical transition.

PubMed

Kramer, Mark A; Truccolo, Wilson; Eden, Uri T; Lepage, Kyle Q; Hochberg, Leigh R; Eskandar, Emad N; Madsen, Joseph R; Lee, Jong W; Maheshwari, Atul; Halgren, Eric; Chu, Catherine J; Cash, Sydney S

2012-12-18

Why seizures spontaneously terminate remains an unanswered fundamental question of epileptology. Here we present evidence that seizures self-terminate via a discontinuous critical transition or bifurcation. We show that human brain electrical activity at various spatial scales exhibits common dynamical signatures of an impending critical transition--slowing, increased correlation, and flickering--in the approach to seizure termination. In contrast, prolonged seizures (status epilepticus) repeatedly approach, but do not cross, the critical transition. To support these results, we implement a computational model that demonstrates that alternative stable attractors, representing the ictal and postictal states, emulate the observed dynamics. These results suggest that self-terminating seizures end through a common dynamical mechanism. This description constrains the specific biophysical mechanisms underlying seizure termination, suggests a dynamical understanding of status epilepticus, and demonstrates an accessible system for studying critical transitions in nature.

Evolutionary origin of the segmental duplication encompassing the wheat GLU-B1 locus encoding the overexpressed Bx7 (Bx7OE) high molecular weight glutenin subunit.

PubMed

Ragupathy, Raja; Naeem, Hamid A; Reimer, Elsa; Lukow, Odean M; Sapirstein, Harry D; Cloutier, Sylvie

2008-01-01

Sequencing of a BAC clone encompassing the Glu-B1 locus in Glenlea, revealed a 10.3 Kb segmental duplication including the Bx7 gene and flanking an LTR retroelement. To better understand the evolution of this locus, two collections of wheat were surveyed. The first consisted of 96 diploid and tetraploid species accessions while the second consisted of 316 Triticum aestivum cultivars and landraces from 41 countries. The genotypes were first characterized by SDS-PAGE and a total of 40 of the 316 T. aestivum accessions were found to display the overexpressed Bx7 phenotype (Bx7OE). Three lines from the 96 diploid/tetraploid collection also displayed the stronger intensity staining characteristic of the Bx7(OE) subunit. The relative amounts of the Bx7 subunit to total HMW-GS were quantified by RP-HPLC for all Bx7OE accessions and a number of checks. The entire collection was assessed for the presence of four DNA markers namely an 18 bp indel of the coding region of Bx7 variant alleles, a 43 bp indel of the 5'-region and the left and right junctions of the LTR retrotransposon borders and the duplicated segment. All 43 accessions found to have the Bx7OE subunit by SDS-PAGE and RP-HPLC produced the four diagnostic PCR amplicons. None of the lines without the Bx7OE had the LTR retroelement/duplication genomic structure. However, the 18 and 43 bp indel were found in accessions other than Bx7OE. These results indicate that the overexpression of the Bx7 HMW-GS is likely the result of a single event, i.e., a gene duplication at the Glu-B1 locus mediated by the insertion of a retroelement. Also, the 18 and 43 bp indels pre-date the duplication event. Allelic variants Bx7*, Bx7 with and without 43 bp insert and Bx7OE were found in both tetraploid and hexaploid collections and shared the same genomic organization. Though the possibility of introgression from T. aestivum to T. turgidum cannot be ruled out, the three structural genomic changes of the B-genome taken together support the hypothesis of multiple polyploidization events involving different tetraploid progenitors.

Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

PubMed Central

Barrick, Doug

2011-01-01

Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

Discordant expression and variable numbers of neighboring GGA- and GAA-rich triplet repeats in the 3' untranslated regions of two groups of messenger RNAs encoded by the rat polymeric immunoglobulin receptor gene.

PubMed Central

Koch, K S; Gleiberman, A S; Aoki, T; Leffert, H L; Feren, A; Jones, A L; Fodor, E J

1995-01-01

An unusual S1-nuclease sensitive microsatellite (STMS) has been found in the single copy, rat polymeric immunoglobulin receptor gene (PIGR) terminal exon. In Fisher rats, elements within or beyond the STMS are expressed variably in the 3' untranslated regions (3'UTRs) of two 'Groups' of PIGR-encoded hepatic mRNAs (pIg-R) during liver regeneration. STMS elements include neighboring constant regions (a 60-bp d[GA]-rich tract with a chi-like octamer, followed by 15 tandem d[GGA] repeats) that merge directly with 36 or 39 tandem d[GAA] repeats (Fisher or Wistar strains, respectively) interrupted by d[AA] between their 5th-6th repeat units. The Wistar STMS is flanked upstream by two regions of nearly contiguous d[CA] or d[CT] repeats in the 3' end of intron 8; and downstream, by a 283 bp 'unit' containing several inversions at its 5' end, and two polyadenylation signals at its 3' end. The 283 nt unit is expressed in Group 1 pIg-R mRNAs; but it is absent in the Group 2 family so that their GAA repeats merge with their poly A tails. In contrast to genomic sequence, GGA triplet repeats are amplified (n > or = 24-26), whereas GAA triplet repeats are truncated variably (n < or = 9-37) and expressed uninterruptedly in both mRNA Groups. These results suggest that 3' end processing of the rat PIGR gene may involve misalignment, slippage and premature termination of RNA polymerase II. The function of this unusual processing and possible roles of chi-like octamers in quiescent or extrahepatic tissues are discussed. Images PMID:7739889

Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

PubMed Central

West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

2005-01-01

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

Resistance to Change and Preference for Variable versus Fixed Response Sequences

ERIC Educational Resources Information Center

Arantes, Joana; Berg, Mark E.; Le, Dien; Grace, Randolph C.

2012-01-01

In Experiment 1, 4 pigeons were trained on a multiple chain schedule in which the initial link was a variable-interval (VI) 20-s schedule signalled by a red or green center key, and terminal links required four responses made to the left (L) and/or right (R) keys. In the REPEAT component, signalled by red keylights, only LRLR terminal-link…

Prenatal diagnosis of hypoplastic left heart syndrome: impact of counseling patterns on parental perceptions and decisions regarding termination of pregnancy.

PubMed

Hilton-Kamm, Debra; Chang, Ruey-Kang; Sklansky, Mark

2012-12-01

An online survey for parents of children with congenital heart disease (CHD) was developed to study parents' experiences at the time of diagnosis. The survey was distributed to online support groups. A total of 841 responses from parents of children with CHD were received during a 4-week period. The current study examined those respondents (211 [25 %]) who reported their child's diagnosis as hypoplastic left heart syndrome (HLHS). Among these, 138 (65 %) reported receiving the diagnosis prenatally. 32 % of those receiving a prenatal diagnosis reported that after they declined to terminate the pregnancy, termination was mentioned again by their physicians. Parents who had termination mentioned again after their initial decline reported significantly lower optimism regarding their child's life expectancy than those who did not have it mentioned again (66 vs. 94 %, p < 0.001); were more likely to interpret the term "rare" to mean "little or no chance of survival" (34 vs. 13 %, p = 0.01); and were more likely to change pediatric cardiologists (PCs) (43 vs. 12 %, p < 0.001). Similarly, 22 % of respondents receiving a prenatal diagnosis reported feeling pressure to terminate the pregnancy by the PC. Those who felt pressure to terminate reported lower optimism about their child's life expectancy than respondents who did not feel pressure (48 vs. 88 %, p < 0.001) and were more likely to choose a new PC (48 vs. 17 %, p < 0.001). In our cohort of parents, when termination of pregnancy was mentioned after the parents declined it, or if the parents felt pressure to terminate, the parents perceived a lower chance of survival, felt less optimistic about their child's life expectancy, and were more likely to choose another PC for long-term follow-up care. Our study could not determine whether repeated discussions of the possibility for termination of pregnancy independently impacts parental optimism regarding prognosis or whether those who counsel with repeated discussions of termination tend to have more guarded notions of the prognosis of children with HLHS. Further study is warranted to identify the implications of counseling patterns on parental perceptions and decisions regarding termination of pregnancy.

Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods.

PubMed

Nakazawa, Yasumoto; Asakura, Tetsuo

2003-06-18

Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.

Mobile genetic elements and cancer. From mutations to gene therapy.

PubMed

Kozeretska, I A; Demydov, S V; Ostapchenko, L I

2011-12-01

In the present review, an association between cancer and the activity of the non-LTR retroelements L1, Alu, and SVA, as well as endogenous retroviruses, in the human genome, is analyzed. Data suggesting that transposons have been involved in embryogenesis and malignization processes, are presented. Events that lead to the activation of mobile elements in mammalian somatic cells, as well as the use of mobile elements in genetic screening and cancer gene therapy, are reviewed.

In vitro characterization of the RS motif in N-terminal head domain of goldfish germinal vesicle lamin B3 necessary for phosphorylation of the p34cdc2 target serine by SRPK1☆

PubMed Central

Yamaguchi, Akihiko; Iwatani, Miho; Ogawa, Mariko; Kitano, Hajime; Matsuyama, Michiya

2013-01-01

The nuclear envelopes surrounding the oocyte germinal vesicles of lower vertebrates (fish and frog) are supported by the lamina, which consists of the protein lamin B3 encoded by a gene found also in birds but lost in the lineage leading to mammals. Like other members of the lamin family, goldfish lamin B3 (gfLB3) contains two putative consensus phosphoacceptor p34cdc2 sites (Ser-28 and Ser-398) for the M-phase kinase to regulate lamin polymerization on the N- and C-terminal regions flanking a central rod domain. Partial phosphorylation of gfLB3 occurs on Ser-28 in the N-terminal head domain in immature oocytes prior to germinal vesicle breakdown, which suggests continual rearrangement of lamins by a novel lamin kinase in fish oocytes. We applied the expression-screening method to isolate lamin kinases by using phosphorylation site Ser-28-specific monoclonal antibody and a vector encoding substrate peptides from a goldfish ovarian cDNA library. As a result, SRPK1 was screened as a prominent lamin kinase candidate. The gfLB3 has a short stretch of the RS repeats (9-SRASTVRSSRRS-20) upstream of the Ser-28, within the N-terminal head. This stretch of repeats is conserved among fish lamin B3 but is not found in other lamins. In vitro phosphorylation studies and GST-pull down assay revealed that SRPK1 bound to the region of sequential RS repeats (9–20) with affinity and recruited serine into the active site by a grab-and-pull manner. These results indicate SRPK1 may phosphorylate the p34cdc2 site in the N-terminal head of GV-lamin B3 at the RS motifs, which have the general property of aggregation. PMID:23772390

A pipeline of programs for collecting and analyzing group II intron retroelement sequences from GenBank

PubMed Central

2013-01-01

Background Accurate and complete identification of mobile elements is a challenging task in the current era of sequencing, given their large numbers and frequent truncations. Group II intron retroelements, which consist of a ribozyme and an intron-encoded protein (IEP), are usually identified in bacterial genomes through their IEP; however, the RNA component that defines the intron boundaries is often difficult to identify because of a lack of strong sequence conservation corresponding to the RNA structure. Compounding the problem of boundary definition is the fact that a majority of group II intron copies in bacteria are truncated. Results Here we present a pipeline of 11 programs that collect and analyze group II intron sequences from GenBank. The pipeline begins with a BLAST search of GenBank using a set of representative group II IEPs as queries. Subsequent steps download the corresponding genomic sequences and flanks, filter out non-group II introns, assign introns to phylogenetic subclasses, filter out incomplete and/or non-functional introns, and assign IEP sequences and RNA boundaries to the full-length introns. In the final step, the redundancy in the data set is reduced by grouping introns into sets of ≥95% identity, with one example sequence chosen to be the representative. Conclusions These programs should be useful for comprehensive identification of group II introns in sequence databases as data continue to rapidly accumulate. PMID:24359548

LINE-1 retrotransposons: from 'parasite' sequences to functional elements.

PubMed

Paço, Ana; Adega, Filomena; Chaves, Raquel

2015-02-01

Long interspersed nuclear elements-1 (LINE-1) are the most abundant and active retrotransposons in the mammalian genomes. Traditionally, the occurrence of LINE-1 sequences in the genome of mammals has been explained by the selfish DNA hypothesis. Nevertheless, recently, it has also been argued that these sequences could play important roles in these genomes, as in the regulation of gene expression, genome modelling and X-chromosome inactivation. The non-random chromosomal distribution is a striking feature of these retroelements that somehow reflects its functionality. In the present study, we have isolated and analysed a fraction of the open reading frame 2 (ORF2) LINE-1 sequence from three rodent species, Cricetus cricetus, Peromyscus eremicus and Praomys tullbergi. Physical mapping of the isolated sequences revealed an interspersed longitudinal AT pattern of distribution along all the chromosomes of the complement in the three genomes. A detailed analysis shows that these sequences are preferentially located in the euchromatic regions, although some signals could be detected in the heterochromatin. In addition, a coincidence between the location of imprinted gene regions (as Xist and Tsix gene regions) and the LINE-1 retroelements was also observed. According to these results, we propose an involvement of LINE-1 sequences in different genomic events as gene imprinting, X-chromosome inactivation and evolution of repetitive sequences located at the heterochromatic regions (e.g. satellite DNA sequences) of the rodents' genomes analysed.

FTIR and FT-Raman spectra and DFT vibrational analysis of phosphorus-containing dendrons

NASA Astrophysics Data System (ADS)

Furer, V. L.; Vandyukova, I. I.; Vandyukov, A. E.; Majoral, J. P.; Caminade, A. M.; Kovalenko, V. I.

2008-12-01

FTIR and FT-Raman spectra of four generations of phosphorus-containing dendrons with terminal aldehyde or P sbnd Cl groups have been recorded and analyzed. Their spectral patterns are determined by the ratio T/ R ( T, the number of terminal groups; R, the number of repeated units). Bands assigned to the core, repeated units and terminal groups were separated by the difference spectroscopy method. The optimized geometry, frequencies and intensity of IR bands of G1v generation dendron with terminal aldehyde groups were obtained by the density functional theory (DFT). It was found that the internal skeleton of molecules exists in a single stable conformation with planar sbnd O- C6H4- CHdbnd N- N( CH3)- P( dbnd S)< fragments, but terminal groups may adopt the t, g, g- and t,- g, g-rotational isomers. The t,- g, g-conformer is 0.74 kcal/mol less stable compared to the t, g, g-conformer. The bond length and bond angles obtained by DFT show the best agreement with experimental data. Relying on DFT calculations a complete assignment of vibrations is proposed for different parts of the studied dendrons. The calculated frequencies and intensity of IR bands of the t, g, g- and t,- g, g-conformers of G1v are found to be in reasonable agreement with the experimental results. The most reactive site in dendron is the core function and vinyl group is preferred for nucleophilic attack. In dendrimer the most reactive are the terminal groups.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

PubMed

Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

2013-02-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

Chromatin tethering effects of hNopp140 are involved in the spatial organization of nucleolus and the rRNA gene transcription

PubMed Central

Tsai, Yi-Tzang; Lin, Chen-I; Chen, Hung-Kai; Lee, Kuo-Ming; Hsu, Chia-Yi; Yang, Shun-Jen

2008-01-01

The short arms of five human acrocentric chromosomes contain ribosomal gene (rDNA) clusters where numerous mini-nucleoli arise at the exit of mitosis. These small nucleoli tend to coalesce into one or a few large nucleoli during interphase by unknown mechanisms. Here, we demonstrate that the N- and C-terminal domains of a nucleolar protein, hNopp140, bound respectively to α-satellite arrays and rDNA clusters of acrocentric chromosomes for nucleolar formation. The central acidic-and-basic repeated domain of hNopp140, possessing a weak self-self interacting ability, was indispensable for hNopp140 to build up a nucleolar round-shaped structure. The N- or the C-terminally truncated hNopp140 caused nucleolar segregation and was able to alter locations of the rDNA transcription, as mediated by detaching the rDNA repeats from the acrocentric α-satellite arrays. Interestingly, an hNopp140 mutant, made by joining the N- and C-terminal domains but excluding the entire central repeated region, induced nucleolar disruption and global chromatin condensation. Furthermore, RNAi knockdown of hNopp140 resulted in dispersion of the rDNA and acrocentric α-satellite sequences away from nucleolus that was accompanied by rDNA transcriptional silence. Our findings indicate that hNopp140, a scaffold protein, is involved in the nucleolar assembly, fusion, and maintenance. PMID:18253863

Sequence of retrovirus provirus resembles that of bacterial transposable elements

NASA Astrophysics Data System (ADS)

Shimotohno, Kunitada; Mizutani, Satoshi; Temin, Howard M.

1980-06-01

The nucleotide sequences of the terminal regions of an infectious integrated retrovirus cloned in the modified λ phage cloning vector Charon 4A have been elucidated. There is a 569-base pair direct repeat at both ends of the viral DNA. The cell-virus junctions at each end consist of a 5-base pair direct repeat of cell DNA next to a 3-base pair inverted repeat of viral DNA. This structure resembles that of a transposable element and is consistent with the protovirus hypothesis that retroviruses evolved from the cell genome.

The Self Primer of the Long Terminal Repeat Retrotransposon Tf1 Is Not Removed during Reverse Transcription

PubMed Central

Atwood-Moore, Angela; Yan, Kenneth; Judson, Robert L.; Levin, Henry L.

2006-01-01

The long terminal repeat retrotransposon Tf1 of Schizosaccharomyces pombe uses a unique mechanism of self priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus-strand synthesis with an 11-nucleotide RNA removed from the 5′ end of its own transcript. We tested whether the self primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self primer. This suggests that integration of the cDNA relies on mechanisms other than reverse transcription to remove the primer. PMID:16873283

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

PubMed

Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

2016-01-01

Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between RipTAL repeats allows for a reconstruction of repeat array biogenesis, for example through slipped strand mispairing or gene conversion. Using these studies we show how RipTALs of broad host range strains evolved convergently toward a shared target sequence. Finally, we discuss the differences between TALE-likes of plant pathogens in the context of disease ecology.

Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm.

PubMed

Nozawa, Kayo; Ishitani, Ryuichiro; Yoshihisa, Tohru; Sato, Mamoru; Arisaka, Fumio; Kanamaru, Shuji; Dohmae, Naoshi; Mangroo, Dev; Senger, Bruno; Becker, Hubert D; Nureki, Osamu

2013-04-01

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.

Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis.

PubMed

Chien, Wenwen; O'Kelly, James; Lu, Daning; Leiter, Amanda; Sohn, Julia; Yin, Dong; Karlan, Beth; Vadgama, Jay; Lyons, Karen M; Koeffler, H Phillip

2011-06-01

Connective tissue growth factor (CTGF/CCN2) belongs to the CCN family of matricellular proteins, comprising Cyr61, CTGF, NovH and WISP1-3. The CCN proteins contain an N-terminal signal peptide followed by four conserved domains sharing sequence similarities with the insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a C-terminal growth factor cysteine knot domain. To investigate the role of CCN2 in breast cancer, we transfected MCF-7 cells with full-length CCN2, and with four mutant constructs in which one of the domains had been deleted. MCF-7 cells stably expressing full-length CCN2 demonstrated reduced cell proliferation, increased migration in Boyden chamber assays and promoted angiogenesis in chorioallantoic membrane assays compared to control cells. Deletion of the C-terminal cysteine knot domain, but not of any other domain-deleted mutants, abolished activities mediated by full-length CCN2. We have dissected the role of CCN2 in breast tumorigenesis on a structural basis.

Linkage map of the fragments of herpesvirus papio DNA.

PubMed Central

Lee, Y S; Tanaka, A; Lau, R Y; Nonoyama, M; Rabin, H

1981-01-01

Herpesvirus papio (HVP), an Epstein-Barr-like virus, causes lymphoblastoid disease in baboons. The physical map of HVP DNA was constructed for the fragments produced by cleavage of HVP DNA with restriction endonucleases EcoRI, HindIII, SalI, and PvuI, which produced 12, 12, 10, and 4 fragments, respectively. The total molecular size of HVP DNA was calculated as close to 110 megadaltons. The following methods were used for construction of the map; (i) fragments near the ends of HVP DNA were identified by treating viral DNA with lambda exonuclease before restriction enzyme digestion; (ii) fragments containing nucleotide sequences in common with fragments from the second enzyme digest of HVP DNA were examined by Southern blot hybridization; and (iii) the location of some fragments was determined by isolating individual fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. Terminal heterogeneity and internal repeats were found to be unique features of HVP DNA molecule. One to five repeats of 0.8 megadaltons were found at both terminal ends. Although the repeats of both ends shared a certain degree of homology, it was not determined whether they were identical repeats. The internal repeat sequence of HVP DNA was found in the EcoRI-C region, which extended from 8.4 to 23 megadaltons from the left end of the molecule. The average number of the repeats was calculated to be seven, and the molecular size was determined to be 1.8 megadaltons. Similar unique features have been reported in EBV DNA (D. Given and E. Kieff, J. Virol. 28:524-542, 1978). Images PMID:6261015

Adenosine-A1 Receptor Agonist Induced Hyperalgesic Priming Type II

PubMed Central

Araldi, Dioneia; Ferrari, Luiz F.; Levine, Jon D.

2016-01-01

We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt) induces a model of the transition to chronic pain that we have termed Type II hyperalgesic priming. Similar to Type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, Type II hyperalgesic priming differs from Type I in being rapidly induced, protein kinase A (PKA), rather than PKCε dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N6-Cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced Type II hyperalgesic priming. In this study we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein αi subunit activation, differently from DAMGO-induced Type II priming, in which it depends on the β/γ subunit. These data implicate a novel form of Gi-protein signaling pathway in the Type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor. PMID:26588695

Protocol for multiple node network

NASA Technical Reports Server (NTRS)

Kirkham, Harold (Inventor)

1995-01-01

The invention is a multiple interconnected network of intelligent message-repeating remote nodes which employs an antibody recognition message termination process performed by all remote nodes and a remote node polling process performed by other nodes which are master units controlling remote nodes in respective zones of the network assigned to respective master nodes. Each remote node repeats only those messages originated in the local zone, to provide isolation among the master nodes.

Protocol for multiple node network

NASA Technical Reports Server (NTRS)

Kirkham, Harold (Inventor)

1994-01-01

The invention is a multiple interconnected network of intelligent message-repeating remote nodes which employs an antibody recognition message termination process performed by all remote nodes and a remote node polling process performed by other nodes which are master units controlling remote nodes in respective zones of the network assigned to respective master nodes. Each remote node repeats only those messages originated in the local zone, to provide isolation among the master nodes.

The armadillo repeat region targets ARVCF to cadherin-based cellular junctions.

PubMed

Kaufmann, U; Zuppinger, C; Waibler, Z; Rudiger, M; Urbich, C; Martin, B; Jockusch, B M; Eppenberger, H; Starzinski-Powitz, A

2000-11-01

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

Adeno-associated virus inverted terminal repeats stimulate gene editing.

PubMed

Hirsch, M L

2015-02-01

Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172

PubMed Central

Zimmerman, Carl-Ulrich R; Rosengarten, Renate; Spergser, Joachim

2011-01-01

Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable ‘UU172 element’ from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses. PMID:21255110

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

PubMed Central

Russo, James J.; Bohenzky, Roy A.; Chien, Ming-Cheng; Chen, Jing; Yan, Ming; Maddalena, Dawn; Parry, J. Preston; Peruzzi, Daniela; Edelman, Isidore S.; Chang, Yuan; Moore, Patrick S.

1996-01-01

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor. PMID:8962146

Analysis of the genome sequence of the pathogenic Muscovy duck parvovirus strain YY reveals a 14-nucleotide-pair deletion in the inverted terminal repeats.

PubMed

Wang, Jianye; Huang, Yu; Zhou, Mingxu; Zhu, Guoqiang

2016-09-01

Genomic information about Muscovy duck parvovirus is still limited. In this study, the genome of the pathogenic MDPV strain YY was sequenced. The full-length genome of YY is 5075 nucleotides (nt) long, 57 nt shorter than that of strain FM. Sequence alignment indicates that the 5' and 3' inverted terminal repeats (ITR) of strain YY contain a 14-nucleotide-pair deletion in the stem of the palindromic hairpin structure in comparison to strain FM and FZ91-30. The deleted region contains one "E-box" site and one repeated motif with the sequence "TTCCGGT" or "ACCGGAA". Phylogenetic trees constructed based the protein coding genes concordantly showed that YY, together with nine other MDPV isolates from various places, clustered in a separate branch, distinct from the branch formed by goose parvovirus (GPV) strains. These results demonstrate that, despite the distinctive deletion, the YY strain still belongs to the classical MDPV group. Moreover, the deletion of ITR may contribute to the genome evolution of MDPV under immunization pressure.

Job Language Performance Requirements for MOS 31M, Multichannel Communications Equipment Operator, Reference Soldier’s Manual Dated 3 March 1978.

DTIC Science & Technology

1978-03-03

TD -204 TD ...radio repeater set retuning ringing equipment signal cables systems lineup j" TA-312/PT TD -204/U i =" " . "J- m~l,. -- ’+- t,,m. + m~l ,,: : J...8217 " "+ . . . . • " + "A Tech cont TD -754/G telephone terminal terminal applicationsterminal lineup test cables voltage checks (p. k .’- - ~ - ~ - ’--

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

PubMed Central

Walker, Sarah E.; Zhou, Fujun; Mitchell, Sarah F.; Larson, Victoria S.; Valasek, Leos; Hinnebusch, Alan G.; Lorsch, Jon R.

2013-01-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment. PMID:23236192

Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)*

PubMed Central

Zhou, Fujun; Walker, Sarah E.; Mitchell, Sarah F.; Lorsch, Jon R.; Hinnebusch, Alan G.

2014-01-01

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC. PMID:24285537

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin.

PubMed

Rostagno, A A; Schwarzbauer, J E; Gold, L I

1999-03-01

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction.

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin.

PubMed Central

Rostagno, A A; Schwarzbauer, J E; Gold, L I

1999-01-01

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction. PMID:10024513

RNA circularization reveals terminal sequence heterogeneity in a double-stranded RNA virus.

PubMed

Widmer, G

1993-03-01

Double-stranded RNA viruses (dsRNA), termed LRV1, have been found in several strains of the protozoan parasite Leishmania. With the aim of constructing a full-length cDNA copy of the viral genome, including its terminal sequences, a protocol based on PCR amplification across the 3'-5' junction of circularized RNA was developed. This method proved to be applicable to dsRNA. It provided a relatively simple alternative to one-sided PCR, without loss of specificity inherent in the use of generic primers. LRV1 terminal nucleotide sequences obtained by this method showed a considerable variation in length, particularly at the 5' end of the positive strand, as well as the potential for forming 3' overhangs. The opposite genomic end terminates in 0, 1, or 2 TCA trinucleotide repeats. These results are compared with terminal sequences derived from one-sided PCR experiments.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

PubMed Central

Parnham, Stuart; Gaines, William A.; Duggan, Brendan M.; Marcotte, William R.

2011-01-01

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all 1H, 13C, and 15N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes. PMID:21152998

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation.

PubMed

Kang, H; Sayner, S L; Gross, K L; Russell, L C; Chinkers, M

2001-09-04

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.

The Extracellular Protein Factor Epf from Streptococcus pyogenes Is a Cell Surface Adhesin That Binds to Cells through an N-terminal Domain Containing a Carbohydrate-binding Module*

PubMed Central

Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H. P.; Whisstock, James C.; Baker, Edward N.; Kreikemeyer, Bernd

2012-01-01

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain. PMID:22977243

The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module.

PubMed

Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H P; Whisstock, James C; Baker, Edward N; Kreikemeyer, Bernd

2012-11-02

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.

Experiences of informational needs and received information following a prenatal diagnosis of congenital heart defect

PubMed Central

Bergman, Gunnar; Wadensten, Barbro; Mattsson, Elisabet

2016-01-01

Abstract Objective To explore the need for information and what information was actually received following prenatal diagnosis of a congenital heart defect, in a country where termination of pregnancy beyond 22 weeks of gestation is not easily possible because of legal constraints. Methods Twenty‐six Swedish‐speaking pregnant women (n = 14) and partners (n = 12) were consecutively recruited for semi‐structured telephone interviews following the prenatal diagnosis of a congenital heart defect. Data were analyzed using content analysis. Results Although high satisfaction with the specialist information was described, the information was considered overwhelming and complex. Objective, honest, and detailed information about multiple subjects were needed, delivered repeatedly, and supplemented by written information/illustrations. Eighteen respondents had used the Internet to search for information and identified issues involving searching difficulties, low quality, and that it was too complex, insufficient, or unspecific. Those who terminated their pregnancy criticized that there was a lack of information about termination of pregnancy, both from health professionals and online sources, resulting in unanswered questions and unpreparedness. Conclusion Individuals faced with a prenatal diagnosis of a congenital heart defect need individualized and repeated information. These needs are not all adequately met, as individuals are satisfied with the specialist consultation but left with unanswered questions regarding pregnancy termination. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. PMID:26991536

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Experiences of informational needs and received information following a prenatal diagnosis of congenital heart defect.

PubMed

Carlsson, Tommy; Bergman, Gunnar; Wadensten, Barbro; Mattsson, Elisabet

2016-06-01

To explore the need for information and what information was actually received following prenatal diagnosis of a congenital heart defect, in a country where termination of pregnancy beyond 22 weeks of gestation is not easily possible because of legal constraints. Twenty-six Swedish-speaking pregnant women (n = 14) and partners (n = 12) were consecutively recruited for semi-structured telephone interviews following the prenatal diagnosis of a congenital heart defect. Data were analyzed using content analysis. Although high satisfaction with the specialist information was described, the information was considered overwhelming and complex. Objective, honest, and detailed information about multiple subjects were needed, delivered repeatedly, and supplemented by written information/illustrations. Eighteen respondents had used the Internet to search for information and identified issues involving searching difficulties, low quality, and that it was too complex, insufficient, or unspecific. Those who terminated their pregnancy criticized that there was a lack of information about termination of pregnancy, both from health professionals and online sources, resulting in unanswered questions and unpreparedness. Individuals faced with a prenatal diagnosis of a congenital heart defect need individualized and repeated information. These needs are not all adequately met, as individuals are satisfied with the specialist consultation but left with unanswered questions regarding pregnancy termination. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Fossil rhabdoviral sequences integrated into arthropod genomes: ontogeny, evolution, and potential functionality.

PubMed

Fort, Philippe; Albertini, Aurélie; Van-Hua, Aurélie; Berthomieu, Arnaud; Roche, Stéphane; Delsuc, Frédéric; Pasteur, Nicole; Capy, Pierre; Gaudin, Yves; Weill, Mylène

2012-01-01

Retroelements represent a considerable fraction of many eukaryotic genomes and are considered major drives for adaptive genetic innovations. Recent discoveries showed that despite not normally using DNA intermediates like retroviruses do, Mononegaviruses (i.e., viruses with nonsegmented, negative-sense RNA genomes) can integrate gene fragments into the genomes of their hosts. This was shown for Bornaviridae and Filoviridae, the sequences of which have been found integrated into the germ line cells of many vertebrate hosts. Here, we show that Rhabdoviridae sequences, the major Mononegavirales family, have integrated only into the genomes of arthropod species. We identified 185 integrated rhabdoviral elements (IREs) coding for nucleoproteins, glycoproteins, or RNA-dependent RNA polymerases; they were mostly found in the genomes of the mosquito Aedes aegypti and the blacklegged tick Ixodes scapularis. Phylogenetic analyses showed that most IREs in A. aegypti derived from multiple independent integration events. Since RNA viruses are submitted to much higher substitution rates as compared with their hosts, IREs thus represent fossil traces of the diversity of extinct Rhabdoviruses. Furthermore, analyses of orthologous IREs in A. aegypti field mosquitoes sampled worldwide identified an integrated polymerase IRE fragment that appeared under purifying selection within several million years, which supports a functional role in the host's biology. These results show that A. aegypti was subjected to repeated Rhabdovirus infectious episodes during its evolution history, which led to the accumulation of many integrated sequences. They also suggest that like retroviruses, integrated rhabdoviral sequences may participate actively in the evolution of their hosts.

25 CFR 11.1114 - Termination.

Code of Federal Regulations, 2014 CFR

2014-04-01

... functions; (iii) The parent(s) has subjected the minor to willful and repeated acts of sexual abuse; (iv) The minor has suffered serious emotional or mental harm due to the act of the parent(s); or (v) The...

25 CFR 11.1114 - Termination.

Code of Federal Regulations, 2013 CFR

2013-04-01

... functions; (iii) The parent(s) has subjected the minor to willful and repeated acts of sexual abuse; (iv) The minor has suffered serious emotional or mental harm due to the act of the parent(s); or (v) The...

25 CFR 11.1114 - Termination.

Code of Federal Regulations, 2012 CFR

2012-04-01

... functions; (iii) The parent(s) has subjected the minor to willful and repeated acts of sexual abuse; (iv) The minor has suffered serious emotional or mental harm due to the act of the parent(s); or (v) The...

25 CFR 11.1114 - Termination.

Code of Federal Regulations, 2011 CFR

2011-04-01

... functions; (iii) The parent(s) has subjected the minor to willful and repeated acts of sexual abuse; (iv) The minor has suffered serious emotional or mental harm due to the act of the parent(s); or (v) The...

Ten tandem repeats of {beta}-hCG 109-118 enhance immunogenicity and anti-tumor effects of {beta}-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

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

Zhang Yankai; Yan Rong; He Yi

2006-07-14

The {beta}-subunit of human chorionic gonadotropin ({beta}-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of {beta}-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with {beta}-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-{beta}hCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residuemore » sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-{beta}hCGCTP37 and HSP65-{beta}hCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-{beta}hCGCTP37 elicited much higher levels of specific anti-{beta}-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-{beta}hCGCTP37, which should suggest that HSP65-X10-{beta}hCGCTP37 may be an effective protein vaccine for the treatment of {beta}-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.« less

The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids. Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts.

PubMed

Jiang, W; Gupta, D; Gallagher, D; Davis, S; Bhavanandan, V P

2000-04-01

We previously elucidated five distinct protein domains (I-V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of approximately 55 tandem repeats of 329 amino acids and that domains III-V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2. 2-q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92-94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68-94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing

PubMed Central

2011-01-01

Background Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models. PMID:21542930

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

PubMed

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

2011-05-04

Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.

Cassandra retrotransposons carry independently transcribed 5S RNA

PubMed Central

Kalendar, Ruslan; Tanskanen, Jaakko; Chang, Wei; Antonius, Kristiina; Sela, Hanan; Peleg, Ofer; Schulman, Alan H.

2008-01-01

We report a group of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous retrotransposons. These elements, named Cassandra, universally carry conserved 5S RNA sequences and associated RNA polymerase (pol) III promoters and terminators in their long terminal repeats (LTRs). They were found in all vascular plants investigated. Uniquely for LTR retrotransposons, Cassandra produces noncapped, polyadenylated transcripts from the 5S pol III promoter. Capped, read-through transcripts containing Cassandra sequences can also be detected in RNA and in EST databases. The predicted Cassandra RNA 5S secondary structures resemble those for cellular 5S rRNA, with high information content specifically in the pol III promoter region. Genic integration sites are common for Cassandra, an unusual feature for abundant retrotransposons. The 5S in each LTR produces a tandem 5S arrangement with an inter-5S spacing resembling that of cellular 5S. The distribution of 5S genes is very variable in flowering plants and may be partially explained by Cassandra activity. Cassandra thus appears both to have adapted a ubiquitous cellular gene for ribosomal RNA for use as a promoter and to parasitize an as-yet-unidentified group of retrotransposons for the proteins needed in its lifecycle. PMID:18408163

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

PubMed

Henderson, Ian R; Liu, Fuquan; Drea, Sinead; Simpson, Gordon G; Dean, Caroline

2005-08-01

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Complete genome sequence of the english isolate of rat cytomegalovirus (Murid herpesvirus 8).

PubMed

Ettinger, Jakob; Geyer, Henriette; Nitsche, Andreas; Zimmermann, Albert; Brune, Wolfram; Sandford, Gordon R; Hayward, Gary S; Voigt, Sebastian

2012-12-01

The complete genome of the English isolate of rat cytomegalovirus (RCMV-E) was determined. RCMV-E has a 202,946-bp genome with noninverting repeats but without terminal repeats. Thus, it differs significantly in size and genomic arrangement from closely related rodent cytomegaloviruses (CMVs). To account for the differences between the rat CMV isolates of Maastricht and England, RCMV-E was classified as Murid herpesvirus 8 by the International Committee on Taxonomy of Viruses.

Synthesis and study of the vibrational spectra of a first generation phosphorus-containing dendrimer with pyridyl functional groups

NASA Astrophysics Data System (ADS)

Furer, V. L.; Vandyukov, A. E.; Tripathi, V.; Majoral, J. P.; Caminade, A. M.; Kovalenko, V. I.

2018-06-01

A new phosphorus-containing dendrimer of the first-generation with potential pharmacological activity was synthesized and studied by spectral methods. The FTIR, FT Raman, 1H and 31P NMR spectra of the first generation dendrimer G1 with a cyclotriphosphazene core, six branches sbnd Osbnd C6H4sbnd CHdbnd Nsbnd N(CH3)sbnd P(S) < and twelve 4-oxyphenethylamidopyridyl end groups sbnd Osbnd C6H4sbnd (CH2)2sbnd NHsbnd COsbnd C5NH4 were recorded. Amide groups of the dendrimer participate in the formation of an intermolecular hydrogen bond. Structure, geometric parameters, the frequency and intensity of the bands in the vibrational spectra were calculated using DFT with PBE functional and TZ2P basis set. Spectral characteristics, charge distribution and reactivity of the core, repeating units and terminal groups of the dendrimer were determined. The first-generation dendrimer molecule has the shape of a concave lens with a slightly non-planar cyclotriphosphazene core and flat repeating units. Repeating units are arranged symmetrically on three on each side of the core, there are no steric hindrances in it and the end groups are able to enter into subsequent reactions and dendrimer has a sufficiently large cavity for accommodating guest molecules. The HOMO covers the repeating units with a noticeable conjugation and the LUMO belongs to the terminal groups.

Parasitism and the retrotransposon life cycle in plants: a hitchhiker's guide to the genome.

PubMed

Sabot, F; Schulman, A H

2006-12-01

LTR (long terminal repeat) retrotransposons are the main components of higher plant genomic DNA. They have shaped their host genomes through insertional mutagenesis and by effects on genome size, gene expression and recombination. These Class I transposable elements are closely related to retroviruses such as the HIV by their structure and presumptive life cycle. However, the retrotransposon life cycle has been closely investigated in few systems. For retroviruses and retrotransposons, individual defective copies can parasitize the activity of functional ones. However, some LTR retrotransposon groups as a whole, such as large retrotransposon derivatives and terminal repeats in miniature, are non-autonomous even though their genomic insertion patterns remain polymorphic between organismal accessions. Here, we examine what is known of the retrotransposon life cycle in plants, and in that context discuss the role of parasitism and complementation between and within retrotransposon groups.

A Transportable VLF/LF Repeater Terminal - A Design Study,

DTIC Science & Technology

1986-07-01

Corporation and marketed commercially as the ROLM 1602B, is expected to be used on the Airborne Command Post (ABNCP) fleet for MMPM processing. The basic...A suitable keyboard/printer for this application is a solid state teletype, the AN/ UGC -120. This device can also function as an AFSATCOM terminal...and as the VLF output device in the event of CPU failure. The AN/ UGC -120 is an Air Force inventory item. 20 INTERSITE COW4UNICATIONS SUBSYSTEM A single

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

PubMed Central

Potter, Kristine D.; Merlino, Natalie M.; Jacobs, Timothy; Gollnick, Paul

2011-01-01

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism controlled by the trp RNA-binding attenuation protein (TRAP). TRAP binds to 11 (G/U)AG repeats in the trp leader transcript and prevents formation of an antiterminator, which allows formation of an intrinsic terminator (attenuator). Previously, formation of the attenuator RNA structure was believed to be solely responsible for signaling RNA polymerase (RNAP) to halt transcription. However, base substitutions that prevent formation of the antiterminator, and thus allow the attenuator structure to form constitutively, do not result in efficient transcription termination. The observation that the attenuator requires the presence of TRAP bound to the nascent RNA to cause efficient transcription termination suggests TRAP has an additional role in causing termination at the attenuator. We show that the trp attenuator is a weak intrinsic terminator due to low GC content of the hairpin stem and interruptions in the U-stretch following the hairpin. We also provide evidence that termination at the trp attenuator requires forward translocation of RNA polymerase and that TRAP binding to the nascent transcript can induce this activity. PMID:21097886

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator.

PubMed

Potter, Kristine D; Merlino, Natalie M; Jacobs, Timothy; Gollnick, Paul

2011-03-01

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism controlled by the trp RNA-binding attenuation protein (TRAP). TRAP binds to 11 (G/U)AG repeats in the trp leader transcript and prevents formation of an antiterminator, which allows formation of an intrinsic terminator (attenuator). Previously, formation of the attenuator RNA structure was believed to be solely responsible for signaling RNA polymerase (RNAP) to halt transcription. However, base substitutions that prevent formation of the antiterminator, and thus allow the attenuator structure to form constitutively, do not result in efficient transcription termination. The observation that the attenuator requires the presence of TRAP bound to the nascent RNA to cause efficient transcription termination suggests TRAP has an additional role in causing termination at the attenuator. We show that the trp attenuator is a weak intrinsic terminator due to low GC content of the hairpin stem and interruptions in the U-stretch following the hairpin. We also provide evidence that termination at the trp attenuator requires forward translocation of RNA polymerase and that TRAP binding to the nascent transcript can induce this activity.

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera)

PubMed Central

Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

2009-01-01

Background Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. Results A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. Conclusion Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation. PMID:19740420

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera).

PubMed

Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

2009-09-09

Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.

Myelodysplastic syndromes and acute myeloid leukemia in cats infected with feline leukemia virus clone33 containing a unique long terminal repeat.

PubMed

Hisasue, Masaharu; Nagashima, Naho; Nishigaki, Kazuo; Fukuzawa, Isao; Ura, Shigeyoshi; Katae, Hiromi; Tsuchiya, Ryo; Yamada, Takatsugu; Hasegawa, Atsuhiko; Tsujimoto, Hajime

2009-03-01

Feline leukemia virus (FeLV) clone33 was obtained from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV from other cats with AML, differs from the LTRs of other known FeLV in that it has 3 tandem direct 47-bp repeats in the upstream region of the enhancer (URE). Here, we injected cats with FeLV clone33 and found 41% developed myelodysplastic syndromes (MDS) characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow. Some of the cats with MDS eventually developed AML. The bone marrow of the majority of cats with FeLV clone33 induced MDS produced fewer erythroid and myeloid colonies upon being cultured with erythropoietin or granulocyte-macrophage colony-stimulating factor (GM-SCF) than bone marrow from normal control cats. Furthermore, the bone marrow of some of the cats expressed high-levels of the apoptosis-related genes TNF-alpha and survivin. Analysis of the proviral sequences obtained from 13 cats with naturally occurring MDS reveal they also bear the characteristic URE repeats seen in the LTR of FeLV clone33 and other proviruses from cats with AML. Deletions and mutations within the enhancer elements are frequently observed in naturally occurring MDS as well as AML. These results suggest that FeLV variants that bear URE repeats in their LTR strongly associate with the induction of both MDS and AML in cats.

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

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

Torella, JP; Boehm, CR; Lienert, F

2013-12-28

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminatormore » parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.« less

Ankyrin repeats of ANKRA2 recognize a PxLPxL motif on the 3M syndrome protein CCDC8.

PubMed

Nie, Jianyun; Xu, Chao; Jin, Jing; Aka, Juliette A; Tempel, Wolfram; Nguyen, Vivian; You, Linya; Weist, Ryan; Min, Jinrong; Pawson, Tony; Yang, Xiang-Jiao

2015-04-07

Peptide motifs are often used for protein-protein interactions. We have recently demonstrated that ankyrin repeats of ANKRA2 and the paralogous bare lymphocyte syndrome transcription factor RFXANK recognize PxLPxL/I motifs shared by megalin, three histone deacetylases, and RFX5. We show here that that CCDC8 is a major partner of ANKRA2 but not RFXANK in cells. The CCDC8 gene is mutated in 3M syndrome, a short-stature disorder with additional facial and skeletal abnormalities. Two other genes mutated in this syndrome encode CUL7 and OBSL1. While CUL7 is a ubiquitin ligase and OBSL1 associates with the cytoskeleton, little is known about CCDC8. Binding and structural analyses reveal that the ankyrin repeats of ANKRA2 recognize a PxLPxL motif at the C-terminal region of CCDC8. The N-terminal part interacts with OBSL1 to form a CUL7 ligase complex. These results link ANKRA2 unexpectedly to 3M syndrome and suggest novel regulatory mechanisms for histone deacetylases and RFX7. Copyright © 2015 Elsevier Ltd. All rights reserved.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

Comparative Genomic Analysis Reveals Multiple Long Terminal Repeats, Lineage-Specific Amplification, and Frequent Interelement Recombination for Cassandra Retrotransposon in Pear (Pyrus bretschneideri Rehd.)

PubMed Central

Yin, Hao; Du, Jianchang; Li, Leiting; Jin, Cong; Fan, Lian; Li, Meng; Wu, Jun; Zhang, Shaoling

2014-01-01

Cassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature (TRIM). Although Cassandra TRIM elements have been found in almost all vascular plants, detailed investigations on the nature, abundance, amplification timeframe, and evolution have not been performed in an individual genome. We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species, including apple, peach, mei, and woodland strawberry. Our data reveal several interesting findings for this particular retrotransposon family: 1) A large number of the intact copies contain three, four, or five long terminal repeats (LTRs) (∼20% in pear); 2) intact copies and solo LTRs with or without target site duplications are both common (∼80% vs. 20%) in each genome; 3) the elements exhibit an overall unbiased distribution among the chromosomes; 4) the elements are most successfully amplified in pear (5,032 copies); and 5) the evolutionary relationships of these elements vary among different lineages, species, and evolutionary time. These results indicate that Cassandra retrotransposons contain more complex structures (elements with multiple LTRs) than what we have known previously, and that frequent interelement unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes. Thus this study provides insights into the property, propensity, and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons, and enhances our understanding of the structural variation, evolutionary history, and transposition process of LTR retrotransposons in plants. PMID:24899073

The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons.

PubMed Central

Xiong, Y; Eickbush, T H

1988-01-01

Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. We present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. We have therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons. Images PMID:2447482

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1.

PubMed

Bustos, Victor H; Ferrarese, Anna; Venerando, Andrea; Marin, Oriano; Allende, Jorge E; Pinna, Lorenzo A

2006-12-26

Multiple phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38-64 of beta-catenin are phosphorylated by CK1 on Ser-45 with low affinity (K(m) approximately 1 mM), whereas intact beta-catenin is phosphorylated at Ser-45 with very high affinity (K(m) approximately 200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70-74 positions or a F74AA mutation in full-length beta-catenin had no significant effect on CK1 phosphorylation efficiency. beta-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131-181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in K(m) value. Implication of the first armadillo repeat in beta-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of beta-catenin from alpha-catenin, further improves CK1 phosphorylation efficiency, lowering the K(m) value to <50 nM, approximating the physiological concentration of beta-catenin. In contrast, alpha-catenin, which interacts with the N-terminal region of beta-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that beta-catenin association with alpha-catenin and beta-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.

The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target

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

Mak, Amanda Nga-Sze; Bradley, Philip; Cernadas, Raul A.

2012-02-10

DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand.more » Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.« less

The Argonaute protein TbAGO1 contributes to large and mini-chromosome segregation and is required for control of RIME retroposons and RHS pseudogene-associated transcripts.

PubMed

Durand-Dubief, Mickaël; Absalon, Sabrina; Menzer, Linda; Ngwabyt, Sandra; Ersfeld, Klaus; Bastin, Philippe

2007-12-01

The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1-/- failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.

The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription.

PubMed Central

Ke, N; Gao, X; Keeney, J B; Boeke, J D; Voytas, D F

1999-01-01

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated. PMID:10411136

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

PubMed Central

Wissing, Silke; Muñoz-Lopez, Martin; Macia, Angela; Yang, Zhiyuan; Montano, Mauricio; Collins, William; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

2012-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs. PMID:21989055

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

PubMed

Raj, Isha; Sadat Al Hosseini, Hamed; Dioguardi, Elisa; Nishimura, Kaoru; Han, Ling; Villa, Alessandra; de Sanctis, Daniele; Jovine, Luca

2017-06-15

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species

PubMed Central

de Hoon, Michiel J. L.; Makita, Yuko; Nakai, Kenta; Miyano, Satoru

2005-01-01

In prokaryotes, genes belonging to the same operon are transcribed in a single mRNA molecule. Transcription starts as the RNA polymerase binds to the promoter and continues until it reaches a transcriptional terminator. Some terminators rely on the presence of the Rho protein, whereas others function independently of Rho. Such Rho-independent terminators consist of an inverted repeat followed by a stretch of thymine residues, allowing us to predict their presence directly from the DNA sequence. Unlike in Escherichia coli, the Rho protein is dispensable in Bacillus subtilis, suggesting a limited role for Rho-dependent termination in this organism and possibly in other Firmicutes. We analyzed 463 experimentally known terminating sequences in B. subtilis and found a decision rule to distinguish Rho-independent transcriptional terminators from non-terminating sequences. The decision rule allowed us to find the boundaries of operons in B. subtilis with a sensitivity and specificity of about 94%. Using the same decision rule, we found an average sensitivity of 94% for 57 bacteria belonging to the Firmicutes phylum, and a considerably lower sensitivity for other bacteria. Our analysis shows that Rho-independent termination is dominant for Firmicutes in general, and that the properties of the transcriptional terminators are conserved. Terminator prediction can be used to reliably predict the operon structure in these organisms, even in the absence of experimentally known operons. Genome-wide predictions of Rho-independent terminators for the 57 Firmicutes are available in the Supporting Information section. PMID:16110342

Fabrication of artificial toroid nanostructures by modified β-sheet peptides.

PubMed

Li, Wen; Li, Jingfang; Lee, Myongsoo

2013-09-25

Facial peptide P1 carrying repeating hydrophobic and hydrophilic residues as well as lysine terminals self-assemble into uniform toroid structures. The sensitive balance between the hydrophobic interactions and electrostatic repulsion dominates the formation of highly curved assemblies.

Repeat induced abortions in Georgia, characteristics of women with multiple pregnancy terminations: secondary analysis of the Reproductive Health Survey 2010.

PubMed

Pestvenidze, Ekaterine; Berdzuli, Nino; Lomia, Nino; Gagua, Tinatin; Umikashvili, Lia; Stray-Pedersen, Babill

2016-10-01

To examine the multi-faceted characteristics of women with repeat induced abortions and assess post-abortion family planning service provision in Georgia. We performed secondary analysis of the data from the Georgian Reproductive Health Survey 2010. A logistic regression model was used to assess the socio-demographic and behavioral factors, contraceptive practices in relation to repeat induced abortions for 2203 women of reproductive age with at least one induced abortion. The Chi-Square test was used to evaluate provision of post-abortion family planning services. Among the targeted women, 70% (n=1539) had repeat induced abortions. The odds of terminating pregnancy raised exponentially with age (OR 3.12, 95% CI: 2.11-4.61), number of complete pregnancies (3 vs. 0-1 complete pregnancies: OR 3.25, 95% CI: 2.36-4.48) and lower education (OR 1.38, 95% CI: 1.10-1.73). The current use of contraception had a protective effect on the occurrence of repeat induced abortions (OR 0.69, 95% CI: 0.53-0.89 for modern and OR 0.68, 95% CI: 0.50-0.92 for traditional methods). The contraceptive counseling and family planning method was provided only to 32% and 6% of post-abortion women, respectively before discharge from the clinic. Repeat induced abortions were found to be significantly more common (P<0.05) among women who did not receive any post-abortion contraceptive at the site of care (n=1627/1929) compared to those who left the abortion facility with family planning method (n=94/125). Low education, higher age, high parity and non-usage of contraceptives carry an increased risk of repeat induced abortions. Post-abortion family planning service delivery is limited in Georgia. Mandating provision of universal post-abortion contraception at the sites of care has a potential to reduce repeat induced abortions and should become a standard of practice for all clinics providing abortion services in Georgia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Structural Analyses of the Ankyrin Repeat Domain of TRPV6 and Related TRPV Ion Channels

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

Phelps, C.B.; Huang, R.J.; Lishko, P.V.

2008-06-03

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third fingermore » loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.« less

Nebulette interacts with filamin C.

PubMed

Holmes, William B; Moncman, Carole L

2008-02-01

The actin-binding proteins, nebulette, and nebulin, are comprised of a four-domain layout containing an acidic N-terminal region, a repeat domain, a serine-rich-linker region, and a Src homology-3 domain. Both proteins contain homologous N-terminal regions that are predicted to be in different environments within the sarcomere. The nebulin acidic N-terminal region is found at the distal ends of the thin filaments. Nebulette, however, is predicted to extend 150 nm from the center of the Z-line. To dissect out the functions of the N-terminal domain of nebulette, we have performed a yeast two-hybrid screen using nebulette residues 1-86 as bait. We have identified filamin-C, ZASP-1, and tropomyosin-1 as binding partners. Characterization of the nebulette-filamin interaction indicates that filamin-C predominantly interacts with the modules. These data suggest that filamin-C, a known component of striated muscle Z-lines, interacts with nebulette modules. Copyright 2007 Wiley-Liss, Inc.

7 CFR 1755.402 - Ground resistance measurements.

Code of Federal Regulations, 2010 CFR

2010-01-01

... ground resistance of electronic equipment such as span line repeaters, carrier terminal equipment... electronic equipment, the ground resistance shall not exceed 25 ohms. Where the measured ground resistance... construction contract may be used. Results of the electronic equipment ground resistance measurements shall be...

7 CFR 1755.402 - Ground resistance measurements.

Code of Federal Regulations, 2011 CFR

2011-01-01

... ground resistance of electronic equipment such as span line repeaters, carrier terminal equipment... electronic equipment, the ground resistance shall not exceed 25 ohms. Where the measured ground resistance... construction contract may be used. Results of the electronic equipment ground resistance measurements shall be...

7 CFR 1755.402 - Ground resistance measurements.

Code of Federal Regulations, 2014 CFR

2014-01-01

... ground resistance of electronic equipment such as span line repeaters, carrier terminal equipment... electronic equipment, the ground resistance shall not exceed 25 ohms. Where the measured ground resistance... construction contract may be used. Results of the electronic equipment ground resistance measurements shall be...

7 CFR 1755.402 - Ground resistance measurements.

Code of Federal Regulations, 2013 CFR

2013-01-01

... ground resistance of electronic equipment such as span line repeaters, carrier terminal equipment... electronic equipment, the ground resistance shall not exceed 25 ohms. Where the measured ground resistance... construction contract may be used. Results of the electronic equipment ground resistance measurements shall be...

7 CFR 1755.402 - Ground resistance measurements.

Code of Federal Regulations, 2012 CFR

2012-01-01

... ground resistance of electronic equipment such as span line repeaters, carrier terminal equipment... electronic equipment, the ground resistance shall not exceed 25 ohms. Where the measured ground resistance... construction contract may be used. Results of the electronic equipment ground resistance measurements shall be...

Copper-silicon-magnesium alloys for latent heat storage

DOE PAGES

Gibbs, P. J.; Withey, E. A.; Coker, E. N.; ...

2016-06-21

The systematic development of microstructure, solidification characteristics, and heat of solidification with composition in copper-silicon-magnesium alloys for thermal energy storage is presented. Differential scanning calorimetry was used to relate the thermal characteristics to microstructural development in the investigated alloys and clarifies the location of one of the terminal three-phase eutectics. Repeated thermal cycling highlights the thermal storage stability of the transformation through multiple melting events. In conclusion, two near-terminal eutectic alloys display high enthalpies of solidification, relatively narrow melting ranges, and stable transformation hysteresis behaviors suited to thermal energy storage.

Comprehensive analysis of all triple helical repeats in beta-spectrins reveals patterns of selective evolutionary conservation.

PubMed

Baines, Anthony J

2003-01-01

The spectrin superfamily (spectrin, alpha-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, alpha and beta chains contain multiple copies of this repeat. beta-spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical beta-spectrins have 17 repeats; beta-heavy spectrins have 30. Here, the repeats of five human beta-spectrins, plus beta-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical beta are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind alpha-spectrin. Repeats 1 of beta-spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of beta-spectrin repeat 1 detects alpha-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.

Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa21[C][W][OA

PubMed Central

Slootweg, Erik J.; Spiridon, Laurentiu N.; Roosien, Jan; Butterbach, Patrick; Pomp, Rikus; Westerhof, Lotte; Wilbers, Ruud; Bakker, Erin; Bakker, Jaap; Petrescu, Andrei-José; Smant, Geert; Goverse, Aska

2013-01-01

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins. PMID:23660837

Orthologs in Arabidopsis thaliana of the Hsp70 interacting protein Hip

PubMed Central

Webb, Mary Alice; Cavaletto, John M.; Klanrit, Preekamol; Thompson, Gary A.

2001-01-01

The Hsp70-interacting protein Hip binds to the adenosine triphosphatase domain of Hsp70, stabilizing it in the adenosine 5′-diphosphate–ligated conformation and promoting binding of target polypeptides. In mammalian cells, Hip is a component of the cytoplasmic chaperone heterocomplex that regulates signal transduction via interaction with hormone receptors and protein kinases. Analysis of the complete genome sequence of the model flowering plant Arabidopsis thaliana revealed 2 genes encoding Hip orthologs. The deduced sequence of AtHip-1 consists of 441 amino acid residues and is 42% identical to human Hip. AtHip-1 contains the same functional domains characterized in mammalian Hip, including an N-terminal dimerization domain, an acidic domain, 3 tetratricopeptide repeats flanked by a highly charged region, a series of degenerate GGMP repeats, and a C-terminal region similar to the Sti1/Hop/p60 protein. The deduced amino acid sequence of AtHip-2 consists of 380 amino acid residues. AtHip-2 consists of a truncated Hip-like domain that is 46% identical to human Hip, followed by a C-terminal domain related to thioredoxin. AtHip-2 is 63% identical to another Hip-thioredoxin protein recently identified in Vitis labrusca (grape). The truncated Hip domain in AtHip-2 includes the amino terminus, the acidic domain, and tetratricopeptide repeats with flanking charged region. Analyses of expressed sequence tag databases indicate that both AtHip-1 and AtHip-2 are expressed in A thaliana and that orthologs of Hip are also expressed widely in other plants. The similarity between AtHip-1 and its mammalian orthologs is consistent with a similar role in plant cells. The sequence of AtHip-2 suggests the possibility of additional unique chaperone functions. PMID:11599566

Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages ▿†

PubMed Central

Born, Yannick; Fieseler, Lars; Marazzi, Janine; Lurz, Rudi; Duffy, Brion; Loessner, Martin J.

2011-01-01

A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen. PMID:21764969

Prevention of status epilepticus-induced brain edema and neuronal cell loss by repeated treatment with high-dose levetiracetam.

PubMed

Itoh, Kouichi; Inamine, Moriyoshi; Oshima, Wataru; Kotani, Masaharu; Chiba, Yoichi; Ueno, Masaki; Ishihara, Yasuhiro

2015-05-22

The management of status epilepticus (SE) is important to prevent mortality and the development of post-SE symptomatic epilepsy. Acquired epilepsy after an initial brain insult by SE can be experimentally reproduced in the murine model of SE induced by pilocarpine. In the present study, we evaluated the possibility of treatment with a high-dose of levetiracetam in this model. Repeated treatment with high-dose levetiracetam after termination of SE by diazepam significantly prevented the incidence of spontaneous recurrent seizures and mortality for at least 28 days. To determine the brain alterations after SE, magnetic resonance imaging was performed. Both T2-weighted imaging and diffusion-weighted imaging showed changes in the limbic regions. These changes in the limbic regions demonstrated the development of cytotoxic edema three hours after SE, followed by the development of vasogenic edema two days after SE. In the pilocarpine-SE model, the incidence of spontaneous recurrent seizures after SE was strongly associated with neuronal damage within a few hours to days after SE by the development of vasogenic edema via the breakdown of the blood-brain barrier in the limbic regions. High-dose levetiracetam significantly suppressed the parameters in the limbic areas. These data indicate that repeated treatment with high-dose levetiracetam for at least two days after SE termination by diazepam is important for controlling the neuronal damage by preventing brain edema. Therefore, these findings suggest that early treatment with high-dose levetiracetam after SE termination by diazepam may protect against adverse sequelae via the inhibition of neurotoxicity induced by brain edema events. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel virulent and broad-host-range Erwinia amylovora bacteriophages reveal a high degree of mosaicism and a relationship to Enterobacteriaceae phages.

PubMed

Born, Yannick; Fieseler, Lars; Marazzi, Janine; Lurz, Rudi; Duffy, Brion; Loessner, Martin J

2011-09-01

A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen.

Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2.

PubMed

Slootweg, Erik J; Spiridon, Laurentiu N; Roosien, Jan; Butterbach, Patrick; Pomp, Rikus; Westerhof, Lotte; Wilbers, Ruud; Bakker, Erin; Bakker, Jaap; Petrescu, Andrei-José; Smant, Geert; Goverse, Aska

2013-07-01

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.

A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3.

PubMed

Loya, Travis J; O'Rourke, Thomas W; Reines, Daniel

2012-08-01

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 'tail' forms an α-helical multimerization domain that helps assemble it onto an RNA substrate.

Analyses of charophyte chloroplast genomes help characterize the ancestral chloroplast genome of land plants.

PubMed

Civaň, Peter; Foster, Peter G; Embley, Martin T; Séneca, Ana; Cox, Cymon J

2014-04-01

Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes.

A High-Density Genetic Map with Array-Based Markers Facilitates Structural and Quantitative Trait Locus Analyses of the Common Wheat Genome

PubMed Central

Iehisa, Julio Cesar Masaru; Ohno, Ryoko; Kimura, Tatsuro; Enoki, Hiroyuki; Nishimura, Satoru; Okamoto, Yuki; Nasuda, Shuhei; Takumi, Shigeo

2014-01-01

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map. PMID:24972598

Analyses of Charophyte Chloroplast Genomes Help Characterize the Ancestral Chloroplast Genome of Land Plants

PubMed Central

Civáň, Peter; Foster, Peter G.; Embley, Martin T.; Séneca, Ana; Cox, Cymon J.

2014-01-01

Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes. PMID:24682153

A high-density genetic map with array-based markers facilitates structural and quantitative trait locus analyses of the common wheat genome.

PubMed

Iehisa, Julio Cesar Masaru; Ohno, Ryoko; Kimura, Tatsuro; Enoki, Hiroyuki; Nishimura, Satoru; Okamoto, Yuki; Nasuda, Shuhei; Takumi, Shigeo

2014-10-01

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1.

PubMed

Vogt, Julia; Wernstedt, Annekatrin; Ripperger, Tim; Pabst, Brigitte; Zschocke, Johannes; Kratz, Christian; Wimmer, Katharina

2016-11-01

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5'-part from the 3'-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms.

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1

PubMed Central

Vogt, Julia; Wernstedt, Annekatrin; Ripperger, Tim; Pabst, Brigitte; Zschocke, Johannes; Kratz, Christian; Wimmer, Katharina

2016-01-01

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5′-part from the 3′-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms. PMID:27329736

Differential chromosomal organization between Saguinus midas and Saguinus bicolor with accumulation of differences the repetitive sequence DNA.

PubMed

Serfaty, Dayane Martins Barbosa; Carvalho, Natália Dayane Moura; Gross, Maria Claudia; Gordo, Marcelo; Schneider, Carlos Henrique

2017-10-01

Saguinus is the largest and most complex genus of the subfamily Callitrichinae, with 23 species distributed from the south of Central America to the north of South America with Saguinus midas having the largest geographical distribution while Saguinus bicolor has a very restricted one, affected by the population expansion in the state of Amazonas. Considering the phylogenetic proximity of the two species along with evidence on the existence of hybrids between them, as well as cytogenetic studies on Saguinus describing a conserved karyotypic macrostructure, we carried out a physical mapping of DNA repeated sequences in the mitotic chromosome of both species, since these sequences are less susceptible to evolutionary pressure and possibly perform an important function in speciation. Both species presented 2n = 46 chromosomes; in S. midas, chromosome Y is the smallest. Multiple ribosomal sites occur in both species, but chromosome pairs three and four may be regarded as markers that differ the species when subjected to G banding and distribution of retroelement LINE 1, suggesting that it may be cytogenetic marker in which it can contribute to identification of first generation hybrids in contact zone. Saguinus bicolor also presented differences in the LINE 1 distribution pattern for sexual chromosome X in individuals from different urban fragments, probably due to geographical isolation. In this context, cytogenetic analyses reveal a differential genomic organization pattern between species S. midas and S. bicolor, in addition to indicating that individuals from different urban fragments have been accumulating differences because of the isolation between them.

Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

PubMed Central

Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

2011-01-01

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

PubMed

Inoue, D; Santiago, P; Horne, W C; Baron, R

1997-10-03

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein

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

York, Joanne; Agnihothram, Sudhakar S.; Romanowski, Victor

2005-12-20

The G2 fusion subunit of the Junin virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins,more » four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that {alpha}-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever.« less

Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein.

PubMed

D'Auria, Giuseppe; Jiménez, Núria; Peris-Bondia, Francesc; Pelaz, Carmen; Latorre, Amparo; Moya, Andrés

2008-01-14

The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity. In contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences.

Antigenic Diversity of the Plasmodium vivax Circumsporozoite Protein in Parasite Isolates of Western Colombia

PubMed Central

Hernández-Martínez, Miguel Ángel; Escalante, Ananías A.; Arévalo-Herrera, Myriam; Herrera, Sócrates

2011-01-01

Circumsporozoite (CS) protein is a malaria antigen involved in sporozoite invasion of hepatocytes, and thus considered to have good vaccine potential. We evaluated the polymorphism of the Plasmodium vivax CS gene in 24 parasite isolates collected from malaria-endemic areas of Colombia. We sequenced 27 alleles, most of which (25/27) corresponded to the VK247 genotype and the remainder to the VK210 type. All VK247 alleles presented a mutation (Gly → Asn) at position 28 in the N-terminal region, whereas the C-terminal presented three insertions: the ANKKAGDAG, which is common in all VK247 isolates; 12 alleles presented the insertion GAGGQAAGGNAANKKAGDAG; and 5 alleles presented the insertion GGNAGGNA. Both repeat regions were polymorphic in gene sequence and size. Sequences coding for B-, T-CD4+, and T-CD8+ cell epitopes were found to be conserved. This study confirms the high polymorphism of the repeat domain and the highly conserved nature of the flanking regions. PMID:21292878

Simian immunodeficiency viruses from African green monkeys display unusual genetic diversity.

PubMed Central

Johnson, P R; Fomsgaard, A; Allan, J; Gravell, M; London, W T; Olmsted, R A; Hirsch, V M

1990-01-01

African green monkeys are asymptomatic carriers of simian immunodeficiency viruses (SIV), commonly called SIVagm. As many as 50% of African green monkeys in the wild may be SIV seropositive. This high seroprevalence rate and the potential for genetic variation of lentiviruses suggested to us that African green monkeys may harbor widely differing genotypes of SIVagm. To investigate this hypothesis, we determined the entire nucleotide sequence of an infectious proviral molecular clone of SIVagm (155-4) and partial sequences (long terminal repeat and Gag) of three other distinct SIVagm isolates (90, gri-1, and ver-1). Comparisons among the SIVagm isolates revealed extreme diversity at the nucleotide and amino acid levels. Long terminal repeat nucleotide sequences varied up to 35% and Gag protein sequences varied up to 30%. The variability among SIVagm isolates exceeded the variability among any other group of primate lentiviruses. Our data suggest that SIVagm has been in the African green monkey population for a long time and may be the oldest primate lentivirus group in existence. PMID:2304139

Porous Cross-Linked Polyimide-Urea Networks

NASA Technical Reports Server (NTRS)

Meador, Mary Ann B. (Inventor); Nguyen, Baochau N. (Inventor)

2015-01-01

Porous cross-linked polyimide-urea networks are provided. The networks comprise a subunit comprising two anhydride end-capped polyamic acid oligomers in direct connection via a urea linkage. The oligomers (a) each comprise a repeating unit of a dianhydride and a diamine and a terminal anhydride group and (b) are formulated with 2 to 15 of the repeating units. The subunit was formed by reaction of the diamine and a diisocyanate to form a diamine-urea linkage-diamine group, followed by reaction of the diamine-urea linkage-diamine group with the dianhydride and the diamine to form the subunit. The subunit has been cross-linked via a cross-linking agent, comprising three or more amine groups, at a balanced stoichiometry of the amine groups to the terminal anhydride groups. The subunit has been chemically imidized to yield the porous cross-linked polyimide-urea network. Also provided are wet gels, aerogels, and thin films comprising the networks, and methods of making the networks.

Porous Cross-Linked Polyimide Networks

NASA Technical Reports Server (NTRS)

Meador, Mary Ann B. (Inventor); Guo, Haiquan (Inventor)

2015-01-01

Porous cross-linked polyimide networks are provided. The networks comprise an anhydride end-capped polyamic acid oligomer. The oligomer (i) comprises a repeating unit of a dianhydride and a diamine and terminal anhydride groups, (ii) has an average degree of polymerization of 10 to 50, (iii) has been cross-linked via a cross-linking agent, comprising three or more amine groups, at a balanced stoichiometry of the amine groups to the terminal anhydride groups, and (iv) has been chemically imidized to yield the porous cross-linked polyimide network. Also provided are porous cross-linked polyimide aerogels comprising a cross-linked and imidized anhydride end-capped polyamic acid oligomer, wherein the oligomer comprises a repeating unit of a dianhydride and a diamine, and the aerogel has a density of 0.10 to 0.333 g/cm.sup.3 and a Young's modulus of 1.7 to 102 MPa. Also provided are thin films comprising aerogels, and methods of making porous cross-linked polyimide networks.

Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.

PubMed

Cho, Uhn Soo; Xu, Wenqing

2007-01-04

Protein phosphatase 2A (PP2A) is a principal Ser/Thr phosphatase, the deregulation of which is associated with multiple human cancers, Alzheimer's disease and increased susceptibility to pathogen infections. How PP2A is structurally organized and functionally regulated remains unclear. Here we report the crystal structure of an AB'C heterotrimeric PP2A holoenzyme. The structure reveals that the HEAT repeats of the scaffold A subunit form a horseshoe-shaped fold, holding the catalytic C and regulatory B' subunits together on the same side. The regulatory B' subunit forms pseudo-HEAT repeats and interacts with the C subunit near the active site, thereby defining substrate specificity. The methylated carboxy-terminal tail of the C subunit interacts with a highly negatively charged region at the interface between A and B' subunits, suggesting that the C-terminal carboxyl methylation of the C subunit promotes B' subunit recruitment by neutralizing charge repulsion. Together, our structural results establish a crucial foundation for understanding PP2A assembly, substrate recruitment and regulation.

Caspase Activity Is Required for Engulfment of Apoptotic Cells

PubMed Central

Shklyar, Boris; Levy-Adam, Flonia; Mishnaevski, Ketty

2013-01-01

Clearance of apoptotic cells by phagocytic neighbors is crucial for normal development of multicellular organisms. However, how phagocytes discriminate between healthy and dying cells remains poorly understood. We focus on glial phagocytosis of apoptotic neurons during development of the Drosophila central nervous system. We identified phosphatidylserine (PS) as a ligand on apoptotic cells for the phagocytic receptor Six Microns Under (SIMU) and report that PS alone is not sufficient for engulfment. Our data reveal that, additionally to PS exposure, caspase activity is required for clearance of apoptotic cells by phagocytes. Here we demonstrate that SIMU recognizes and binds PS on apoptotic cells through its N-terminal EMILIN (EMI), Nimrod 1 (NIM1), and NIM2 repeats, whereas the C-terminal NIM3 and NIM4 repeats control SIMU affinity to PS. Based on the structure-function analysis of SIMU, we discovered a novel mechanism of internal inhibition responsible for differential affinities of SIMU to its ligand which might prevent elimination of living cells exposing PS on their surfaces. PMID:23754750

The UNC-45 Myosin Chaperone: From Worms to Flies to Vertebrates

PubMed Central

Lee, Chi F.; Melkani, Girish C.; Bernstein, Sanford I.

2014-01-01

UNC-45 is a UCS domain protein that is critical for myosin stability and function. It likely aides in folding myosin during cellular differentiation and maintenance and protects myosin from denaturation during stress. Invertebrates have a single unc-45 gene that is expressed in both muscle and non-muscle tissues. Vertebrates possess one gene expressed in striated muscle (unc-45b) and one that is more generally expressed (unc-45a). Structurally, UNC-45 is composed of a series of alpha-helices connected by loops. It has an N-terminal TPR domain that binds to Hsp90 and a central domain composed of armadillo repeats. Its C-terminal UCS domain, which is also comprised of helical armadillo repeats, interacts with myosin. In this review, we present biochemical, structural and genetic analyses of UNC-45 in Caenorhabditis elegans, Drosophila melanogaster and various vertebrates. Further, we provide insights into UNC-45 functions, its potential mechanism of action and its roles in human disease. PMID:25376491

Lack of detection of feline leukemia and feline sarcoma viruses in diffuse iris melanomas of cats by immunohistochemistry and polymerase chain reaction.

PubMed

Cullen, Cheryl L; Haines, Deborah M; Jackson, Marion L; Grahn, Bruce H

2002-07-01

Diffuse iris melanoma was confirmed by light-microscopic examination in 10 formalin-fixed, paraffin-embedded globes from 10 cats. To determine if feline leukemia virus or a replication defective feline leukemia virus, feline sarcoma virus, was present in these anterior uveal melanomas, immunohistochemistry and polymerase chain reaction for feline leukemia virus were utilized. Immunohistochemical staining for feline leukemia virus glycoprotein 70 was performed on all 10 tumors using an avidin-biotin complex technique. The DNA was extracted from each specimen and a 166-base pair region of the feline leukemia virus long terminal repeat was targeted by polymerase chain reaction. Immunohistochemical staining for feline leukemia virus glycoprotein 70 and polymerase chain reaction amplification of a feline leukemia virus long terminal repeat region were negative in all cases. Feline leukemia virus/feline sarcoma virus was not detected in any neoplasms and therefore was unlikely to play a role in the tumorigenesis of these feline diffuse iris melanomas.

The repeating nucleotide sequence in the repetitive mitochondrial DNA from a "low-density" petite mutant of yeast.

PubMed Central

Van Kreijl, C F; Bos, J L

1977-01-01

The repeating nucleotide sequence of 68 base pairs in the mtDNA from an ethidium-induced cytoplasmic petite mutant of yeast has been determined. For sequence analysis specifically primed and terminated RNA copies, obtained by in vitro transcription of the separated strands, were use. The sequence consists of 66 consecutive AT base pairs flanked by two GC pairs and comprises nearly all of the mutant mitochondrial genome. The sequence, moreover, also represents the first part of wild-type mtDNA sequence so far. Images PMID:198740

Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs

PubMed Central

Morita, Teppei; Nishino, Ryo; Aiba, Hiroji

2017-01-01

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3′ end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs. PMID:28606943

Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes

PubMed Central

Lee, Michael; Hills, Mark; Conomos, Dimitri; Stutz, Michael D.; Dagg, Rebecca A.; Lau, Loretta M.S.; Reddel, Roger R.; Pickett, Hilda A.

2014-01-01

Telomeres are terminal repetitive DNA sequences on chromosomes, and are considered to comprise almost exclusively hexameric TTAGGG repeats. We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. This indicates that telomerase is most likely an error-prone reverse transcriptase that misincorporates nucleotides at specific positions on the telomerase RNA template. In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres. PMID:24225324

[Direct human DNA damage by unfavorable environmental and climatic factors].

PubMed

Doroshtuk, N A; Postnov, A Iu; Doroshtuk, A D; Khasanova, E B; Konovalova, N V; Khesuani, Iu D; Osiaeva, M K; Rodnenkov, O V; Chazova, I E

2014-01-01

To study the impact of simulated climatic conditions of the 2010 summer in Moscow on the telomere repeats of chromosomes in human blood cells. The climatic conditions of July-August 2010 in Moscow were simulated at the Medical Technical Complex, Institute of Biomedical Problems, Russian Academy of Sciences. The relative length of the telomeric repeats of blood cell chromosomes from 6 apparently healthy volunteers was measured by quantitative real-time polymerase chain reaction. These conditions were ascertained to lead to a statistically significant decline in the length of telomere repeats in the terminal portions of chromosomes by 15%. Environmental changes and abnormal temperature rises may result in oxidative stress accompanied by telomere shortening, which can be, in turn, a factor of premature aging.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

PubMed Central

2017-01-01

ABSTRACT Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy. PMID:29046446

Three Groups of Transposable Elements with Contrasting Copy Number Dynamics and Host Responses in the Maize (Zea mays ssp. mays) Genome

PubMed Central

Diez, Concepcion M.; Meca, Esteban; Tenaillon, Maud I.; Gaut, Brandon S.

2014-01-01

Most angiosperm nuclear DNA is repetitive and derived from silenced transposable elements (TEs). TE silencing requires substantial resources from the plant host, including the production of small interfering RNAs (siRNAs). Thus, the interaction between TEs and siRNAs is a critical aspect of both the function and the evolution of plant genomes. Yet the co-evolutionary dynamics between these two entities remain poorly characterized. Here we studied the organization of TEs within the maize (Zea mays ssp mays) genome, documenting that TEs fall within three groups based on the class and copy numbers. These groups included DNA elements, low copy RNA elements and higher copy RNA elements. The three groups varied statistically in characteristics that included length, location, age, siRNA expression and 24∶22 nucleotide (nt) siRNA targeting ratios. In addition, the low copy retroelements encompassed a set of TEs that had previously been shown to decrease expression within a 24 nt siRNA biogenesis mutant (mop1). To investigate the evolutionary dynamics of the three groups, we estimated their abundance in two landraces, one with a genome similar in size to that of the maize reference and the other with a 30% larger genome. For all three accessions, we assessed TE abundance as well as 22 nt and 24 nt siRNA content within leaves. The high copy number retroelements are under targeted similarly by siRNAs among accessions, appear to be born of a rapid bust of activity, and may be currently transpositionally dead or limited. In contrast, the lower copy number group of retrolements are targeted more dynamically and have had a long and ongoing history of transposition in the maize genome. PMID:24743518

Nuclear Exclusion of the HIV-1 host defense factor APOBEC3G requires a novel cytoplasmic retention signal and is not dependent on RNA binding.

PubMed

Bennett, Ryan P; Presnyak, Vladimir; Wedekind, Joseph E; Smith, Harold C

2008-03-21

Human APOBEC3G (hA3G) is a host factor that defends against HIV-1 as well as other exogenous retroviruses and endogenous retroelements. To this end, hA3G is restricted to the cytoplasm of T lymphocytes where it interacts with viral RNA and proteins to assemble with viral particles causing a post-entry block during reverse transcription. hA3G also exhibits a mechanism to inhibit the reverse transcription of retroelements by RNA binding and sequestration into mRNA processing centers in the cytoplasm. We have determined that the molecular basis for this specialized property of hA3G is a novel cytoplasmic retention signal (CRS) that is necessary and sufficient to restrict wild-type hA3G and chimeric constructs to the cytoplasm. The CRS resides within amino acids 113-128 and is embedded within a basic flanking sequence and does not require RNA binding to retain hA3G in the cytoplasm. Paralogs of hA3G that have nuclear or cytoplasmic distributions differ from hA3G within the region encompassing the CRS motif with respect to charge and amino acid composition. We propose that the CRS enables hA3G to interact with cytoplasmic factors, and thereby enables hA3G to serve in host cell defense by restricting an antiviral sentinel to the cytoplasm. The CRS lies in a region involved in both Gag and Vif interactions; therefore, identification of this motif has important implications for the design of therapeutics that target HIV-1 while maintaining antiviral and cellular functions.

Identification of a Retroelement from the Resurrection Plant Boea hygrometrica That Confers Osmotic and Alkaline Tolerance in Arabidopsis thaliana

PubMed Central

Shen, Chun-Ying; Xu, Guang-Hui; Chen, Shi-Xuan; Song, Li-Zhen; Li, Mei-Jing; Wang, Li-Li; Zhu, Yan; Lv, Wei-Tao; Gong, Zhi-Zhong; Liu, Chun-Ming; Deng, Xin

2014-01-01

Functional genomic elements, including transposable elements, small RNAs and non-coding RNAs, are involved in regulation of gene expression in response to plant stress. To identify genomic elements that regulate dehydration and alkaline tolerance in Boea hygrometrica, a resurrection plant that inhabits drought and alkaline Karst areas, a genomic DNA library from B. hygrometrica was constructed and subsequently transformed into Arabidopsis using binary bacterial artificial chromosome (BIBAC) vectors. Transgenic lines were screened under osmotic and alkaline conditions, leading to the identification of Clone L1-4 that conferred osmotic and alkaline tolerance. Sequence analyses revealed that L1-4 contained a 49-kb retroelement fragment from B. hygrometrica, of which only a truncated sequence was present in L1-4 transgenic Arabidopsis plants. Additional subcloning revealed that activity resided in a 2-kb sequence, designated Osmotic and Alkaline Resistance 1 (OAR1). In addition, transgenic Arabidopsis lines carrying an OAR1-homologue also showed similar stress tolerance phenotypes. Physiological and molecular analyses demonstrated that OAR1-transgenic plants exhibited improved photochemical efficiency and membrane integrity and biomarker gene expression under both osmotic and alkaline stresses. Short transcripts that originated from OAR1 were increased under stress conditions in both B. hygrometrica and Arabidopsis carrying OAR1. The relative copy number of OAR1 was stable in transgenic Arabidopsis under stress but increased in B. hygrometrica. Taken together, our results indicated a potential role of OAR1 element in plant tolerance to osmotic and alkaline stresses, and verified the feasibility of the BIBAC transformation technique to identify functional genomic elements from physiological model species. PMID:24851859

Chompy: an infestation of MITE-like repetitive elements in the crocodilian genome.

PubMed

Ray, David A; Hedges, Dale J; Herke, Scott W; Fowlkes, Justin D; Barnes, Erin W; LaVie, Daniel K; Goodwin, Lindsey M; Densmore, Llewellyn D; Batzer, Mark A

2005-12-05

Interspersed repeats are a major component of most eukaryotic genomes and have an impact on genome size and stability, but the repetitive element landscape of crocodilian genomes has not yet been fully investigated. In this report, we provide the first detailed characterization of an interspersed repeat element in any crocodilian genome. Chompy is a putative miniature inverted-repeat transposable element (MITE) family initially recovered from the genome of Alligator mississippiensis (American alligator) but also present in the genomes of Crocodylus moreletii (Morelet's crocodile) and Gavialis gangeticus (Indian gharial). The element has all of the hallmarks of MITEs including terminal inverted repeats, possible target site duplications, and a tendency to form secondary structures. We estimate the copy number in the alligator genome to be approximately 46,000 copies. As a result of their size and unique properties, Chompy elements may provide a useful source of genomic variation for crocodilian comparative genomics.

NMR Analysis of Amide Hydrogen Exchange Rates in a Pentapeptide-Repeat Protein from A. thaliana.

PubMed

Xu, Shenyuan; Ni, Shuisong; Kennedy, Michael A

2017-05-23

At2g44920 from Arabidopsis thaliana is a pentapeptide-repeat protein (PRP) composed of 25 repeats capped by N- and C-terminal α-helices. PRP structures are dominated by four-sided right-handed β-helices typically consisting of mixtures of type II and type IV β-turns. PRPs adopt repeated five-residue (Rfr) folds with an Rfr consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Unlike other PRPs, At2g44920 consists exclusively of type II β-turns. At2g44920 is predicted to be located in the thylakoid lumen although its biochemical function remains unknown. Given its unusual structure, we investigated the biophysical properties of At2g44920 as a representative of the β-helix family to determine if it had exceptional global stability, backbone dynamics, or amide hydrogen exchange rates. Circular dichroism measurements yielded a melting point of 62.8°C, indicating unexceptional global thermal stability. Nuclear spin relaxation measurements indicated that the Rfr-fold core was rigid with order parameters ranging from 0.7 to 0.9. At2g44920 exhibited a striking range of amide hydrogen exchange rates spanning 10 orders of magnitude, with lifetimes ranging from minutes to several months. A weak correlation was found among hydrogen exchange rates, hydrogen bonding energies, and amino acid solvent-accessible areas. Analysis of contributions from fast (approximately picosecond to nanosecond) backbone dynamics to amide hydrogen exchange rates revealed that the average order parameter of amides undergoing fast exchange was significantly smaller compared to those undergoing slow exchange. Importantly, the activation energies for amide hydrogen exchange were found to be generally higher for the slowest exchanging amides in the central Rfr coil and decreased toward the terminal coils. This could be explained by assuming that the concerted motions of two preceding or following coils required for hydrogen bond disruption and amide hydrogen exchange have a higher activation energy compared to that required for displacement of a single coil to facilitate amide hydrogen exchange in either the terminal or penultimate coils. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

PubMed

Verdyck, Pieter; Berckmoes, Veerle; De Vos, Anick; Verpoest, Willem; Liebaers, Inge; Bonduelle, Maryse; De Rycke, Martine

2015-10-01

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS. © 2015 Wiley Periodicals, Inc.

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

PubMed Central

Yang, Pinfen; Sale, Winfield S.

1998-01-01

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo. PMID:9843573

Evidence that a proposed repeated segment of glutamine residues is expressed in the Huntington disease protein

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

Jou, Y.S.; Myers, R.M.

1994-09-01

Huntington disease (HD) appears to be caused by a mutation that results in an expanded number of CAG repeats at the 5{prime} end of the gene. The nucleotide sequence of the gene and cDNA clones predicts a 347 kd protein that contains a stretch of polyglutamine, encoded by the CAG repeat, located 17 amino acids downstream from the proposed translation initiation site. Because understanding the mechanisms of the pathology of HD depends on whether the CAG-repeat is expressed in the protein, we used antibodies directed against portions of the predicted HD gene product to probe the structure of the proteinmore » in tissue culture cells. Two peptides, one located amino-terminal to the proposed polyglutamine stretch (hd1 peptide FESLKSFQQ from amino acids 11-19) and one located in the carboxy-terminal half of the predicted protein (hd2 peptide QQPRNKPLK from amino acids 2531-2539), were used to elicit polyclonal antibodies in NZW rabbits. We affinity-purified the antibodies and used them to analyze the HD protein. Both antisera specifically recognize the peptides used to elicit them, as well as the appropriate portions of the HD protein expressed in E. coli. Western blot analysis showed that both antisera recognize a protein with an apparent molecular weight of approximately 350,000 in human, monkey, rat and mouse cell lines, including two neutronal cell lines. These results, in combination with immunoprecipitation experiments, suggest strongly that the proposed polyglutamine stretch is indeed translated in the HD protein and is evolutionarily conserved in various mammalian species.« less

Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae

PubMed Central

Erkes, Annett; Reschke, Maik; Boch, Jens

2017-01-01

Abstract Transcription activator-like effectors (TALEs) are secreted by plant–pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity. PMID:28637323

A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3

PubMed Central

Loya, Travis J.; O’Rourke, Thomas W.; Reines, Daniel

2012-01-01

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene. Transcription terminates via the Nrd1-Nab3-Sen1 complex and is degraded by the nuclear exosome. Using a sensitive terminator read-through assay, we identified trans-acting Terminator Override (TOV) genes that operate this terminator. Four genes were identified: the RNA polymerase II phosphatase SSU72, the RNA polymerase II binding protein PCF11, the TRAMP subunit TRF4 and the hnRNP-like, NAB3. The TOV phenotype can be explained by the loss of function of these gene products as described in models in which termination and RNA degradation are coupled to the phosphorylation state of RNA polymerase II's repeat domain. The most interesting mutations were those found in NAB3, which led to the finding that the removal of merely three carboxy-terminal amino acids compromised Nab3's function. This region of previously unknown function is distant from the protein's well-known RNA binding and Nrd1 binding domains. Structural homology modeling suggests this Nab3 ‘tail’ forms an α-helical multimerization domain that helps assemble it onto an RNA substrate. PMID:22564898

Neural Stem Cell Delivery of Therapeutic Antibodies to Treat Breast Cancer Brain Metastases

DTIC Science & Technology

2009-10-01

brain tumors remains dismal. High-grade neoplasms , such as gliomas, are highly invasive and spawn widely disseminated microsatellites that have... myeloproliferative sarcoma virus long terminal repeat negative control region deleted (MND promoter), allows suffi- cient expression in some cell types at a level

Maxi CAI with a Micro.

ERIC Educational Resources Information Center

Gerhold, George; And Others

This paper describes an effective microprocessor-based CAI system which has been repeatedly tested by a large number of students and edited accordingly. Tasks not suitable for microprocessor based systems (authoring, testing, and debugging) were handled on larger multi-terminal systems. This approach requires that the CAI language used on the…

Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

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

Sun, Ping; Tropea, Joseph E.; Austin, Brian P.

2008-05-03

The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonasmore » aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.« less

The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine

DOE PAGES

Julier, Ziad; Martino, Mikaël M.; de Titta, Alexandre; ...

2015-02-24

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with itsmore » neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. We reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8 + T cell responses in two murine cancer models.« less

Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs.

PubMed

Morita, Teppei; Nishino, Ryo; Aiba, Hiroji

2017-09-01

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3' end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs. © 2017 Morita et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Spectroscopic and molecular structure investigation of the phosphorus-containing G‧2 dendrimer with terminal aldehyde groups using DFT method

NASA Astrophysics Data System (ADS)

Furer, V. L.; Vandyukov, A. E.; Majoral, J. P.; Caminade, A. M.; Kovalenko, V. I.

2015-02-01

The FTIR and FT Raman spectra of the second generation dendrimer G‧2 built from thiophosphoryl core with terminal aldehyde groups have been recorded. The structural optimization and normal mode analysis were performed for model compound C, consisting of thiophosphoryl core, one branch with three repeated units, and four 4-oxybenzaldehyde terminal groups on the basis of the density functional theory (DFT) at the PBE/TZ2P level. The vibrational frequencies, infrared and Raman intensities for the t,g,g- and t,-g,g-conformers of the terminal groups were calculated. The t,g,g-conformer is 2.0 kcal/mol less stable compared to t,-g,g-conformer. A reliable assignment of the fundamental bands observed in the experimental IR and Raman spectra of dendrimer was achieved. For the low generations (G‧1 to G‧3) the disk form of studied dendrimer molecules is the most probable. For higher generations, the shape of dendrimer molecules will be that of a cauliflower.

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

PubMed Central

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

PubMed

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L; Salt, Jennifer N; Goring, Daphne R

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

Genome Dynamics and Evolution of the Mla (Powdery Mildew) Resistance Locus in BarleyW⃞

PubMed Central

Wei, Fusheng; Wing, Rod A.; Wise, Roger P.

2002-01-01

Genes that confer defense against pathogens often are clustered in the genome and evolve via diverse mechanisms. To evaluate the organization and content of a major defense gene complex in cereals, we determined the complete sequence of a 261-kb BAC contig from barley cv Morex that spans the Mla (powdery mildew) resistance locus. Among the 32 predicted genes on this contig, 15 are associated with plant defense responses; 6 of these are associated with defense responses to powdery mildew disease but function in different signaling pathways. The Mla region is organized as three gene-rich islands separated by two nested complexes of transposable elements and a 45-kb gene-poor region. A heterochromatic-like region is positioned directly proximal to Mla and is composed of a gene-poor core with 17 families of diverse tandem repeats that overlap a hypermethylated, but transcriptionally active, gene-dense island. Paleontology analysis of long terminal repeat retrotransposons indicates that the present Mla region evolved over a period of >7 million years through a variety of duplication, inversion, and transposon-insertion events. Sequence-based recombination estimates indicate that R genes positioned adjacent to nested long terminal repeat retrotransposons, such as Mla, do not favor recombination as a means of diversification. We present a model for the evolution of the Mla region that encompasses several emerging features of large cereal genomes. PMID:12172030

Characterization of gene encoding amylopullulanase from plant-originated lactic acid bacterium, Lactobacillus plantarum L137.

PubMed

Kim, Jong-Hyun; Sunako, Michihiro; Ono, Hisayo; Murooka, Yoshikatsu; Fukusaki, Eiichiro; Yamashita, Mitsuo

2008-11-01

A starch-hydrolyzing lactic acid bacterium, Lactobacillus plantarum L137, was isolated from traditional fermented food made from fish and rice in the Philippines. A gene (apuA) encoding an amylolytic enzyme from Lactobacillus plantarum L137 was cloned, and its nucleotide sequence was determined. The apuA gene consisted of an open reading frame of 6171 bp encoding a protein of 2056 amino acids, the molecular mass of which was calculated to be 215,625 Da. The catalytic domains of amylase and pullulanase were located in the same region within the middle of the N-terminal region. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the N-terminal region, a six-amino-acid sequence (Asp-Ala/Thr-Ala-Asn-Ser-Thr) is repeated 39 times, and a three-amino-acid sequence (Gln-Pro-Thr) is repeated 50 times in the C-terminal region. The apuA gene was subcloned in L. plantarum NCL21, which is a plasmid-cured derivative of the wild-type L137 strain and has no amylopullulanase activity, and the gene was overexpressed under the control of its own promoter. The ApuA enzyme from this recombinant L. plantarum NCL21 harboring apuA gene was purified. The enzyme has both alpha-amylase and pullulanase activities. The N-terminal sequence of the purified enzyme showed that the signal peptide was cleaved at Ala(36) and the molecular mass of the mature extracellular enzyme is 211,537 Da. The major reaction products from soluble starch were maltotriose (G3) and maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. From these results, we concluded that ApuA is an amylolytic enzyme belonging to the amylopullulanase family.

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II.

PubMed

Chang, A; Cheang, S; Espanel, X; Sudol, M

2000-07-07

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.

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

T Yeh; C Lee; L Amzel

Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both inmore » Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.« less

Moral dilemmas of women undergoing pregnancy termination for medical reasons in Poland.

PubMed

Zaręba, Kornelia; Ciebiera, Michał; Bińkowska, Małgorzata; Jakiel, Grzegorz

2017-08-01

We explored the religious views and dilemmas of Polish women making the decision to terminate a pregnancy. The article discusses the highly restrictive legislation and significant influence of the Church on the lives of Polish citizens. This study was designed to investigate the effect of religious and political beliefs, social and moral conditioning and professional support on the decision to abort a fetus. A 65-item questionnaire was administered to 60 participants at the time of their pregnancy termination. Pregnancy termination was performed outside the resident county in 32% of cases. Approximately 88% of respondents declared themselves Catholic, but only 22% intended to admit to the pregnancy termination during confession. Five percent of respondents feared the reaction of the priest, while the remaining respondents did not perceive termination of pregnancy for medical reasons as a sin. Of the women who had previously opposed pregnancy termination, 27% changed their mind once they were personally involved. The decision to abort a pregnancy for medical reasons is sensitive to religious and social determinants, especially in the current political situation in which abortion may become prohibited in Poland. The high response rate (100%) was probably the result of the patients' attitudes: they repeatedly emphasised they were thankful for the help and empathy of the medical personnel and for being allowed to undergo the procedure. In Poland, the majority of centres use conscience clauses to justify their refusal to terminate a pregnancy.

Genetic diversity of Danthonia spicata (L.) Beauv. Based on genomic simple sequence repeat markers

USDA-ARS?s Scientific Manuscript database

Danthonia spicata, commonly known as poverty oatgrass, is a perennial bunch-type grass native to North America. D. spicata has dimorphic seed heads; the hypothesis is that terminal seed heads allow some level of outcrossing and axial seed heads are only self-fertilized. However, there is no genetic ...

Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation

PubMed Central

Cohen, Todd J.; Constance, Brian H.; Hwang, Andrew W.; James, Michael; Yuan, Chao-Xing

2016-01-01

Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer’s disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies. PMID:27383765

Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

PubMed

Cohen, Todd J; Constance, Brian H; Hwang, Andrew W; James, Michael; Yuan, Chao-Xing

2016-01-01

Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

Paternal and sibling incest: a case report.

PubMed

Celbis, Osman; Ozcan, M Erkan; Ozdemir, Bora

2006-01-01

A case is reported of a female victim of paternal incest, who had also been raped repeatedly by her elder brother for two years. A survey of the literature showed no other report of such a case from Turkey. This does not necessarily mean that the incidence of paternal and sibling incest does not happen, but may indicate that incestuous abuse is not reported or handled without making it known to legal authorities. The victim was first raped by her 16 year-old brother when she was 9 years old. He raped her repeatedly over a period of two years, until he left home. Her father began raping the victim when she was 13 year-old, leaving her pregnant at age 15. He took her to a doctor for a termination of pregnancy. The father continued abuse after the termination. The victim left home to marry a man. The father filed a lawsuit against the man for taking the victim away from home. More openness and awareness of incest in Turkey may encourage the victims to seek help from medical and legal authorities.

Large diversity of the piggyBac-like elements in the genome of Tribolium castaneum

PubMed Central

Wang, Jianjun; Du, Yuzhou; Wang, Suzhi; Brown, Sue; Park, Yoonseong

2011-01-01

The piggyBac transposable element, originally discovered in the cabbage looper, Trichoplusia ni, has been widely used in insect transgenesis including the red flour beetle Tribolium castaneum. We surveyed piggyBac-like (PLE) sequences in the genome of Tribolium castaneum by homology searches using as queries the diverse PLE sequences that have been described previously. The search yielded a total of 32 piggyBac-like elements (TcPLEs) which were classified into 14 distinct groups. Most of the TcPLEs contain defective functional motifs in that they are lacking inverted terminal repeats or have disrupted open reading frames. Only one single copy of TcPLE1 appears to be intact with imperfect 16 bp inverted terminal repeats flanking an open reading frame encoding a transposase of 571 amino acid residues. Many copies of TcPLEs were found to be inserted into or close to other transposon-like sequences. This large diversity of TcPLEs with generally low copy numbers suggests multiple invasions of the TcPLEs over a long evolutionary time without extensive multiplications or occurrence of rapid loss of TcPLEs copies. PMID:18342253

Discovery and analysis of an active long terminal repeat-retrotransposable element in Aspergillus oryzae.

PubMed

Jie Jin, Feng; Hara, Seiichi; Sato, Atsushi; Koyama, Yasuji

2014-01-01

Wild-type Aspergillus oryzae RIB40 contains two copies of the AO090005001597 gene. We previously constructed A. oryzae RIB40 strain, RKuAF8B, with multiple chromosomal deletions, in which the AO090005001597 copy number was found to be increased significantly. Sequence analysis indicated that AO090005001597 is part of a putative 6,000-bp retrotransposable element, flanked by two long terminal repeats (LTRs) of 669 bp, with characteristics of retroviruses and retrotransposons, and thus designated AoLTR (A. oryzae LTR-retrotransposable element). AoLTR comprised putative reverse transcriptase, RNase H, and integrase domains. The deduced amino acid sequence alignment of AoLTR showed 94% overall identity with AFLAV, an A. flavus Tf1/sushi retrotransposon. Quantitative real-time RT-PCR showed that AoLTR gene expression was significantly increased in the RKuAF8B, in accordance with the increased copy number. Inverse PCR indicated that the full-length retrotransposable element was randomly integrated into multiple genomic locations. However, no obvious phenotypic changes were associated with the increased AoLTR gene copy number.

Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection.

PubMed

Wang, Chao; Li, Xue; Yu, Fei; Lu, Lu; Jiang, Xifeng; Xu, Xiaoyu; Wang, Huixin; Lai, Wenqing; Zhang, Tianhong; Zhang, Zhenqing; Ye, Ling; Jiang, Shibo; Liu, Keliang

2016-08-26

Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection.

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

PubMed Central

Petrova, Olga A; Mantsyzov, Alexey B; Rodina, Elena V; Efimov, Sergey V; Hackenberg, Claudia; Hakanpää, Johanna; Klochkov, Vladimir V; Lebedev, Andrej A; Chugunova, Anastasia A; Malyavko, Alexander N; Zatsepin, Timofei S; Mishin, Alexey V; Zvereva, Maria I

2018-01-01

Abstract The elongation of single-stranded DNA repeats at the 3′-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids—telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the ‘fork’ at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis. PMID:29294091

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome

PubMed Central

De Nicola, Beatrice; Lech, Christopher J.; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N.; Phan, Anh Tuân

2016-01-01

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. PMID:27298260

Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

NASA Astrophysics Data System (ADS)

Sun, Xiao-Hong; Baltimore, David

1989-04-01

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

Female choice reveals terminal investment in male mealworm beetles, Tenebrio molitor, after a repeated activation of the immune system.

PubMed

Krams, I; Daukšte, J; Kivleniece, I; Krama, T; Rantala, M J; Ramey, G; Šauša, L

2011-01-01

Increasing evidence suggests that secondary sexual traits reflect immunocompetence of males in many animal species. This study experimentally investigated whether a parasite-like immunological challenge via a nylon implant affects sexual attractiveness of males in Tenebrio molitor L. (Coleoptera: Tenebrionidae) Although a single immunological challenge significantly reduced sexual attractiveness and locomotor activity of males, it had no adverse effect on their survival. A second immune challenge of the same males increased their attractiveness. However, it was found that the repeated challenge significantly reduced locomotor activity of males and caused higher mortality. This result indicates terminal investment on sexual signaling, which is supposedly based on a trade-off between pheromone production and energy expenditures needed for such activities as recovery of immune system and locomotor activity. When the third implantation was carried out in the same group of males, melanization of nylon implants was found to be lower in more attractive than in less attractive males. This suggests that males that became sexually attractive after the second immune challenge did not invest in recovery of their immune system.

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

PubMed

Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

2001-06-01

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

Structural Basis of PP2A Inhibition by Small t Antigen

PubMed Central

Cho, Uhn Soo; Morrone, Seamus; Sablina, Anna A; Arroyo, Jason D; Hahn, William C; Xu, Wenqing

2007-01-01

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST. PMID:17608567

Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection

PubMed Central

Wang, Chao; Li, Xue; Yu, Fei; Lu, Lu; Jiang, Xifeng; Xu, Xiaoyu; Wang, Huixin; Lai, Wenqing; Zhang, Tianhong; Zhang, Zhenqing; Ye, Ling; Jiang, Shibo; Liu, Keliang

2016-01-01

Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection. PMID:27562370

Female Choice Reveals Terminal Investment in Male Mealworm Beetles, Tenebrio molitor, after a Repeated Activation of the Immune System

PubMed Central

Krams, I; Daukšte, J; Kivleniece, I; Krama, T; Rantala, MJ; Ramey, G; Šauša, L

2011-01-01

Increasing evidence suggests that secondary sexual traits reflect immunocompetence of males in many animal species. This study experimentally investigated whether a parasite-like immunological challenge via a nylon implant affects sexual attractiveness of males in Tenebrio molitor L. (Coleoptera: Tenebrionidae) Although a single immunological challenge significantly reduced sexual attractiveness and locomotor activity of males, it had no adverse effect on their survival. A second immune challenge of the same males increased their attractiveness. However, it was found that the repeated challenge significantly reduced locomotor activity of males and caused higher mortality. This result indicates terminal investment on sexual signaling, which is supposedly based on a trade-off between pheromone production and energy expenditures needed for such activities as recovery of immune system and locomotor activity. When the third implantation was carried out in the same group of males, melanization of nylon implants was found to be lower in more attractive than in less attractive males. This suggests that males that became sexually attractive after the second immune challenge did not invest in recovery of their immune system. PMID:21864151

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

PubMed

Ray, Hind; Moreau, Karen; Dizin, Eva; Callebaut, Isabelle; Venezia, Nicole Dalla

2006-06-16

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

A Legionella pneumophila collagen-like protein encoded by a gene with a variable number of tandem repeats is involved in the adherence and invasion of host cells.

PubMed

Vandersmissen, Liesbeth; De Buck, Emmy; Saels, Veerle; Coil, David A; Anné, Jozef

2010-05-01

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen and the causative agent of Legionnaires' disease, a severe pneumonia in humans. Analysis of the Legionella sequenced genomes revealed a gene with a variable number of tandem repeats (VNTRs), whose number varies between strains. We examined the strain distribution of this gene among a collection of 108 clinical, environmental and hot spring serotype I strains. Twelve variants were identified, but no correlation was observed between the number of repeat units and clinical and environmental strains. The encoded protein contains the C-terminal consensus motif of outer membrane proteins and has a large region of collagen-like repeats that is encoded by the VNTR region. We have therefore annotated this protein Lcl for Legionella collagen-like protein. Lcl was shown to contribute to the adherence and invasion of host cells and it was demonstrated that the number of repeat units present in lcl had an influence on these adhesion characteristics.

Functions of the 3′ and 5′ genome RNA regions of members of the genus Flavivirus

PubMed Central

Brinton, Margo A.; Basu, Mausumi

2015-01-01

The positive sense genomes of members of the genus Flavivirus in the family Flaviviridae are ~11 kb nts in length and have a 5′ type I cap but no 3′ poly A. The 5′ and 3′ terminal regions contain short conserved sequences that are proposed to be repeated remnants of an ancient sequence. However, the functions of most of these conserved sequences have not yet been determined. The terminal regions of the genome also contain multiple conserved RNA structures. Functional data for many of these structures has been obtained. Three sets of complementary 3′ and 5′ terminal region sequences, some of which are located in conserved RNA structures, interact to form a panhandle structure that is required for initiation of minus strand RNA synthesis with the 5′ terminal structure functioning as the promoter. How the switch from the terminal RNA structure base pairing to the long distance RNA-RNA interaction is triggered and regulated is not well understood but evidence suggests involvement of a cell protein binding to three sites on the 3′ terminal RNA structures and a cis-acting metastable 3′ RNA element in the 3′ terminal structure. Cell proteins may also be involved in facilitating exponential replication of nascent genomic RNA within replication vesicles at later times of infection cycle. Other conserved RNA structures and/or sequences in the 5′ and 3′ terminal regions have been proposed to regulate genome translation. Additional functions of the 5′ and 3′ terminal sequences have also been reported. PMID:25683510

Two tandemly repeated telomere-associated sequences in Nicotiana plumbaginifolia.

PubMed

Chen, C M; Wang, C T; Wang, C J; Ho, C H; Kao, Y Y; Chen, C C

1997-12-01

Two tandemly repeated telomere-associated sequences, NP3R and NP4R, have been isolated from Nicotiana plumbaginifolia. The length of a repeating unit for NP3R and NP4R is 165 and 180 nucleotides respectively. The abundance of NP3R, NP4R and telomeric repeats is, respectively, 8.4 x 10(4), 6 x 10(3) and 1.5 x 10(6) copies per haploid genome of N. plumbaginifolia. Fluorescence in situ hybridization revealed that NP3R is located at the ends and/or in interstitial regions of all 10 chromosomes and NP4R on the terminal regions of three chromosomes in the haploid genome of N. plumbaginifolia. Sequence homology search revealed that not only are NP3R and NP4R homologous to HRS60 and GRS, respectively, two tandem repeats isolated from N. tabacum, but that NP3R and NP4R are also related to each other, suggesting that they originated from a common ancestral sequence. The role of these repeated sequences in chromosome healing is discussed based on the observation that two to three copies of a telomere-similar sequence were present in each repeating unit of NP3R and NP4R.

Condensin loaded onto the replication fork barrier site in the rRNA gene repeats during S phase in a FOB1-dependent fashion to prevent contraction of a long repetitive array in Saccharomyces cerevisiae.

PubMed

Johzuka, Katsuki; Terasawa, Masahiro; Ogawa, Hideyuki; Ogawa, Tomoko; Horiuchi, Takashi

2006-03-01

An average of 200 copies of the rRNA gene (rDNA) is clustered in a long tandem array in Saccharomyces cerevisiae. FOB1 is known to be required for expansion/contraction of the repeats by stimulating recombination, thereby contributing to the maintenance of the average copy number. In Deltafob1 cells, the repeats are still maintained without any fluctuation in the copy number, suggesting that another, unknown system acts to prevent repeat contraction. Here, we show that condensin acts together with FOB1 in a functionally complemented fashion to maintain the long tandem repeats. Six condensin mutants possessing severely contracted rDNA repeats were isolated in Deltafob1 cells but not in FOB1+ cells. We also found that the condensin complex associated with the nontranscribed spacer region of rDNA with a major peak coincided with the replication fork barrier (RFB) site in a FOB1-dependent fashion. Surprisingly, condensin association with the RFB site was established during S phase and was maintained until anaphase. These results indicate that FOB1 plays a novel role in preventing repeat contraction by regulating condensin association and suggest a link between replication termination and chromosome condensation and segregation.

Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins.

PubMed

Cortajarena, Aitziber L; Kajander, Tommi; Pan, Weilan; Cocco, Melanie J; Regan, Lynne

2004-04-01

Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein-peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.

Adenovirus sequences required for replication in vivo.

PubMed Central

Wang, K; Pearson, G D

1985-01-01

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I. Images PMID:2991857

Distinct retroelement classes define evolutionary breakpoints demarcating sites of evolutionary novelty

PubMed Central

Longo, Mark S; Carone, Dawn M; Green, Eric D; O'Neill, Michael J; O'Neill, Rachel J

2009-01-01

Background Large-scale genome rearrangements brought about by chromosome breaks underlie numerous inherited diseases, initiate or promote many cancers and are also associated with karyotype diversification during species evolution. Recent research has shown that these breakpoints are nonrandomly distributed throughout the mammalian genome and many, termed "evolutionary breakpoints" (EB), are specific genomic locations that are "reused" during karyotypic evolution. When the phylogenetic trajectory of orthologous chromosome segments is considered, many of these EB are coincident with ancient centromere activity as well as new centromere formation. While EB have been characterized as repeat-rich regions, it has not been determined whether specific sequences have been retained during evolution that would indicate previous centromere activity or a propensity for new centromere formation. Likewise, the conservation of specific sequence motifs or classes at EBs among divergent mammalian taxa has not been determined. Results To define conserved sequence features of EBs associated with centromere evolution, we performed comparative sequence analysis of more than 4.8 Mb within the tammar wallaby, Macropus eugenii, derived from centromeric regions (CEN), euchromatic regions (EU), and an evolutionary breakpoint (EB) that has undergone convergent breakpoint reuse and past centromere activity in marsupials. We found a dramatic enrichment for long interspersed nucleotide elements (LINE1s) and endogenous retroviruses (ERVs) and a depletion of short interspersed nucleotide elements (SINEs) shared between CEN and EBs. We analyzed the orthologous human EB (14q32.33), known to be associated with translocations in many cancers including multiple myelomas and plasma cell leukemias, and found a conserved distribution of similar repetitive elements. Conclusion Our data indicate that EBs tracked within the class Mammalia harbor sequence features retained since the divergence of marsupials and eutherians that may have predisposed these genomic regions to large-scale chromosomal instability. PMID:19630942

The Ma Gene for Complete-Spectrum Resistance to Meloidogyne Species in Prunus Is a TNL with a Huge Repeated C-Terminal Post-LRR Region1[C][W

PubMed Central

Claverie, Michel; Dirlewanger, Elisabeth; Bosselut, Nathalie; Van Ghelder, Cyril; Voisin, Roger; Kleinhentz, Marc; Lafargue, Bernard; Abad, Pierre; Rosso, Marie-Noëlle; Chalhoub, Boulos; Esmenjaud, Daniel

2011-01-01

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1–TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling. PMID:21482634

Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

PubMed Central

Verma, Subhash C.; Lu, Jie; Cai, Qiliang; Kosiyatrakul, Settapong; McDowell, Maria E.; Schildkraut, Carl L.; Robertson, Erle S.

2011-01-01

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences. PMID:22072974

U3 long terminal repeat-mediated induction of intracellular immunity by a murine retrovirus: a novel model of latency for retroviruses.

PubMed Central

Gorska-Flipot, I; Huang, M; Cantin, M; Rassart, E; Massé, G; Jolicoeur, P

1992-01-01

BL/VL3 radiation leukemia virus (RadLV) is a thymotropic, highly leukemogenic murine leukemia virus (MuLV) which is unable to replicate in vitro in mouse fibroblasts. We have previously reported that the U3 long terminal repeat region of its genome is responsible for this block (E. Rassart, Y. Paquette, and P. Jolicoeur, J. Virol. 62:3840-3848, 1988). By using hybrids of permissive and resistant cells infected with BL/VL3 RadLV or fibrotropic MuLV, we found that the resistant phenotype was dominant. Investigation to determine at which step of the virus cycle the block operates revealed that integration, transcription, and translation of the BL/VL3 viral genome occurred at normal levels in nonpermissive cells. The BL/VL3 RadLV Pr65gag proteins made in nonpermissive cells were also myristylated and located at the membrane, and the levels of their cleaved products were similar to those of fibrotropic MuLV. However, processing of BL/VL3 RadLV Pr85env was impaired in nonpermissive cells. Virions were not released into the culture medium of nonpermissive cells, as measured by reverse transcriptase activity and by content in p30 or gp70 protein and as documented by lower levels of budding particles seen by electron microscopy. These results indicate that BL/VL3 RadLV replication is blocked at a late stage of the virus cycle, i.e., at virion assembly. Interestingly, these BL/VL3 RadLV-infected nonpermissive fibroblasts were resistant to superinfection by fibrotropic Moloney MuLV, and this resistance also occurred at a late step of the Moloney virus cycle. Since this block is dominant, it appears that the U3 long terminal repeat region of the BL/VL3 viral genome has the ability to induce a cellular suppressor factor(s), thus bringing intracellular immunity against itself and against other ecotropic MuLVs. Images PMID:1433513

Genomic Landscape of Long Terminal Repeat Retrotransposons (LTR-RTs) and Solo LTRs as Shaped by Ectopic Recombination in Chicken and Zebra Finch.

PubMed

Ji, Yanzhu; DeWoody, J Andrew

2016-06-01

Transposable elements (TEs) are nearly ubiquitous among eukaryotic genomes, but TE contents vary dramatically among phylogenetic lineages. Several mechanisms have been proposed as drivers of TE dynamics in genomes, including the fixation/loss of a particular TE insertion by selection or drift as well as structural changes in the genome due to mutation (e.g., recombination). In particular, recombination can have a significant and directional effect on the genomic TE landscape. For example, ectopic recombination removes internal regions of long terminal repeat retrotransposons (LTR-RTs) as well as one long terminal repeat (LTR), resulting in a solo LTR. In this study, we focus on the intra-species dynamics of LTR-RTs and solo LTRs in bird genomes. The distribution of LTR-RTs and solo LTRs in birds is intriguing because avian recombination rates vary widely within a given genome. We used published linkage maps and whole genome assemblies to study the relationship between recombination rates and LTR-removal events in the chicken and zebra finch. We hypothesized that regions with low recombination rates would harbor more full-length LTR-RTs (and fewer solo LTRs) than regions with high recombination rates. We tested this hypothesis by comparing the ratio of full-length LTR-RTs and solo LTRs across chromosomes, across non-overlapping megabase windows, and across physical features (i.e., centromeres and telomeres). The chicken data statistically supported the hypothesis that recombination rates are inversely correlated with the ratio of full-length to solo LTRs at both the chromosome level and in 1-Mb non-overlapping windows. We also found that the ratio of full-length to solo LTRs near chicken telomeres was significantly lower than those ratios near centromeres. Our results suggest a potential role of ectopic recombination in shaping the chicken LTR-RT genomic landscape.

Terminal sequence importance of de novo proteins from binary-patterned library: stable artificial proteins with 11- or 12-amino acid alphabet.

PubMed

Okura, Hiromichi; Takahashi, Tsuyoshi; Mihara, Hisakazu

2012-06-01

Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

The Termination of Checking and the Role of Just Right Feelings: A Study of Obsessional Checkers Compared with Anxious and Non-clinical Controls.

PubMed

Salkovskis, Paul M; Millar, Josie; Gregory, James D; Wahl, Karina

2017-03-01

Repeated checking in OCD can be understood from a cognitive perspective as the motivated need to achieve certainty about the outcome of a potentially risky action, leading to the application of Elevated Evidence Requirements (EER) and overuse of subjective criteria. Twenty-four obsessional checkers, 22 anxious controls, and 26 non-clinical controls were interviewed about and rated recent episodes where they felt (a) they needed to check and (b) checked mainly out of habit (i.e. not obsessionally). Both subjective and objective criteria were rated as significantly more important in obsessional checkers than in controls; obsessional checkers also used more criteria overall for the termination of the check, and rated more criteria as "extremely important" than the control groups. The termination of the check was rated as more effortful for obsessional checkers than for the comparison groups. Analysis of the interview data was consistent with the ratings. Feelings of "rightness" were associated with the termination of a check for obsessional checkers but not for controls. Results were consistent with the proposal that the use of "just right feelings" to terminate checking are related to EER.

N-terminal nesprin-2 variants regulate β-catenin signalling

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

Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa

2016-07-15

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragmentmore » of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.« less

Molecular cloning and sequence analysis of the gene coding for the 57kDa soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

USGS Publications Warehouse

Chien, Maw-Sheng; Gilbert , Teresa L.; Huang, Chienjin; Landolt, Marsha L.; O'Hara, Patrick J.; Winton, James R.

1992-01-01

The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated Mr value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of Mr 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

Interplay between I308 and Y310 residues in the third repeat of microtubule-binding domain is essential for tau filament formation.

PubMed

Naruto, Keiko; Minoura, Katsuhiko; Okuda, Ryouhei; Taniguchi, Taizo; In, Yasuko; Ishida, Toshimasa; Tomoo, Koji

2010-10-08

Investigation of the mechanism of tau polymerization is indispensable for finding inhibitory conditions or identifying compounds preventing the formation of paired helical filament or oligomers. Tau contains a microtubule-binding domain consisting of three or four repeats in its C-terminal half. It has been considered that the key event in tau polymerization is the formation of a β-sheet structure arising from a short hexapeptide (306)VQIVYK(311) in the third repeat of tau. In this paper, we report for the first time that the C-H⋯π interaction between Ile308 and Tyr310 is the elemental structural scaffold essential for forming a dry "steric zipper" structure in tau amyloid fibrils. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

PubMed

Gachon, F; Thebault, S; Peleraux, A; Devaux, C; Mesnard, J M

2000-05-01

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

The Carboxy-Terminal Domain of Erb1 Is a Seven-Bladed ß-Propeller that Binds RNA

PubMed Central

Marcin, Wegrecki; Neira, Jose Luis; Bravo, Jeronimo

2015-01-01

Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis. PMID:25880847

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

PubMed Central

Wang, Xianzhe; Maynard, Jennifer A.

2015-01-01

The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

Molecular Interactions Involved in the Transactivation of the Human T-Cell Leukemia Virus Type 1 Promoter Mediated by Tax and CREB-2 (ATF-4)

PubMed Central

Gachon, Frederic; Thebault, Sabine; Peleraux, Annick; Devaux, Christian; Mesnard, Jean-Michel

2000-01-01

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation. PMID:10779337

Structure and evolution of N-domains in AAA metalloproteases.

PubMed

Scharfenberg, Franka; Serek-Heuberger, Justyna; Coles, Murray; Hartmann, Marcus D; Habeck, Michael; Martin, Jörg; Lupas, Andrei N; Alva, Vikram

2015-02-27

Metalloproteases of the AAA (ATPases associated with various cellular activities) family play a crucial role in protein quality control within the cytoplasmic membrane of bacteria and the inner membrane of eukaryotic organelles. These membrane-anchored hexameric enzymes are composed of an N-terminal domain with one or two transmembrane helices, a central AAA ATPase module, and a C-terminal Zn(2+)-dependent protease. While the latter two domains have been well studied, so far, little is known about the N-terminal regions. Here, in an extensive bioinformatic and structural analysis, we identified three major, non-homologous groups of N-domains in AAA metalloproteases. By far, the largest one is the FtsH-like group of bacteria and eukaryotic organelles. The other two groups are specific to Yme1: one found in plants, fungi, and basal metazoans and the other one found exclusively in animals. Using NMR and crystallography, we determined the subunit structure and hexameric assembly of Escherichia coli FtsH-N, exhibiting an unusual α+β fold, and the conserved part of fungal Yme1-N from Saccharomyces cerevisiae, revealing a tetratricopeptide repeat fold. Our bioinformatic analysis showed that, uniquely among these proteins, the N-domain of Yme1 from the cnidarian Hydra vulgaris contains both the tetratricopeptide repeat region seen in basal metazoans and a region of homology to the N-domains of animals. Thus, it is a modern-day representative of an intermediate in the evolution of animal Yme1 from basal eukaryotic precursors. Copyright © 2015. Published by Elsevier Ltd.

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

Identification of a non-LTR retrotransposon from the gypsy moth

Treesearch

K.J. Garner; J.M. Slavicek

1999-01-01

A family of highly repetitive elements, named LDT1, has been identified in the gypsy moth, Lymantria dispar. The complete element is 5.4 kb in length and lacks long-terminal repeats, The element contains two open reading frames with a significant amino acid sequence similarity to several non-LTR retrotransposons. The first open reading frame contains...

Protective efficacy of a recombinant bacterial artificial chromosome clone of a very virulent Marek’s disease virus containing a reticuloendotheliosis virus long terminal repeat

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV), an alphaherpesvirus, causes Marek’s disease (MD), a lymphoproliferative disease in poultry characterized by T-cell lymphomas, nerve lesions and mortality. Vaccination is used worldwide to control MD, but increasingly virulent field strains can overcome this protection, d...

Direct and Distal Effects of Noncontingent Juice on Rumination Exhibited by a Child with Autism

ERIC Educational Resources Information Center

Kliebert, Megan L.; Tiger, Jeffrey H.

2011-01-01

Previous research has demonstrated the efficacy of the noncontingent delivery of foods and liquids at suppressing rumination, the repeated regurgitation and rechewing of partially digested food. However, it is unclear how long this reduction is maintained after caregivers terminate this procedure. The current study examined the direct and distal…

The investigation of Ag/ZnO interface system by first principle: The structural, electronic and optical properties

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

Cheng, Hai-Xia; Wang, Xiao-Xu; Beijing Computing Center, Beijing 100094

Ag/ZnO interfaces have been investigated for both of Zn-termination and O-termination by the first principle based on density functional theory. Our calculations demonstrate that the Ag atoms go inward from the Ag/ZnO interface, and the Zn and O atoms are all move outward bulk in the Zn-termination interface, and the changes are just opposite for O-termination. These behaviors are in agreement with the other studies in literatures. Furthermore, an expansion situation is observed in the first two Zn-O bilayer and first three Ag monolayers for both of Zn-termination and O-termination interfaces by comparing with the pure ZnO(0001) and Ag(111) surfaces.more » Moreover, the valence-band both of O-2p and Zn-3d states of Ag/ZnO interface gradual close to Femi level as the Zn, O atoms locate at the deeper layer for Zn-termination, but it is the other way round for O-termination. Calculated absorption spectrum indicates that the absorption intensity of Zn-termination interface is stronger than that of O-termination in the lower energy range (visible light region). These properties of ZnO surfaces are also evaluated for comparison with interfaces. - Graphical abstract: The structures of Ag/ZnO interface: Zn-termination (left) and O-termination (right). In this Ag/ZnO interface system, the ZnO (0001) surface is rotated 30°(R30), and Ag (111) surface is built (2×2) supercell, then a (2×√3) R30 Ag/ZnO interface is constructed using the supercell method (i.e. periodically repeated slabs). The lattice mismatch of (2×√3) R30 Ag/ZnO (2.6% mismatch) is smaller than that of (1×1) Ag/ZnO (11% mismatch).« less

The Big Bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups.

PubMed

Koonin, Eugene V; Wolf, Yuri I; Nagasaki, Keizo; Dolja, Valerian V

2008-12-01

The recent discovery of RNA viruses in diverse unicellular eukaryotes and developments in evolutionary genomics have provided the means for addressing the origin of eukaryotic RNA viruses. The phylogenetic analyses of RNA polymerases and helicases presented in this Analysis article reveal close evolutionary relationships between RNA viruses infecting hosts from the Chromalveolate and Excavate supergroups and distinct families of picorna-like viruses of plants and animals. Thus, diversification of picorna-like viruses probably occurred in a 'Big Bang' concomitant with key events of eukaryogenesis. The origins of the conserved genes of picorna-like viruses are traced to likely ancestors including bacterial group II retroelements, the family of HtrA proteases and DNA bacteriophages.

Suppressor of sable [Su(s)] and Wdr82 down-regulate RNA from heat-shock-inducible repetitive elements by a mechanism that involves transcription termination

PubMed Central

Brewer-Jensen, Paul; Wilson, Carrie B.; Abernethy, John; Mollison, Lonna; Card, Samantha

2016-01-01

Although RNA polymerase II (Pol II) productively transcribes very long genes in vivo, transcription through extragenic sequences often terminates in the promoter-proximal region and the nascent RNA is degraded. Mechanisms that induce early termination and RNA degradation are not well understood in multicellular organisms. Here, we present evidence that the suppressor of sable [su(s)] regulatory pathway of Drosophila melanogaster plays a role in this process. We previously showed that Su(s) promotes exosome-mediated degradation of transcripts from endogenous repeated elements at an Hsp70 locus (Hsp70-αβ elements). In this report, we identify Wdr82 as a component of this process and show that it works with Su(s) to inhibit Pol II elongation through Hsp70-αβ elements. Furthermore, we show that the unstable transcripts produced during this process are polyadenylated at heterogeneous sites that lack canonical polyadenylation signals. We define two distinct regions that mediate this regulation. These results indicate that the Su(s) pathway promotes RNA degradation and transcription termination through a novel mechanism. PMID:26577379

Perceived Personal Control Buffers Terminal Decline in Well-Being

PubMed Central

Gerstorf, Denis; Heckhausen, Jutta; Ram, Nilam; Infurna, Frank J.; Schupp, Juergen; Wagner, Gert G.

2015-01-01

Recent research has repeatedly demonstrated that well-being typically evinces precipitous deterioration close to the end of life. However, the determinants of individual differences in these terminal declines are note well understood. In this study, we examine the role of perceived personal control as a potential buffer against steep terminal declines in well-being. We applied single- and multi-phase growth models to up to 25-year longitudinal data from 1,641 now deceased participants of the national German Socio-Economic Panel Study (SOEP; age at death: M = 74 years; SD = 14; 49% women). Results revealed that perceiving more personal control over one’s life was related to subsequently higher late-life well-being, less severe rates of late-life declines, and a later onset of terminal decline. Associations were independent of key predictors of mortality, including age, gender, SES, and disability. These findings suggest that feeling in control may ameliorate steep end-of-life decline in well-being. We also discuss scenarios for when and how processes of goal disengagement and giving up control may become beneficial. PMID:25244480

The partly Aalen's model for recurrent event data with a dependent terminal event.

PubMed

Chen, Chyong-Mei; Shen, Pao-Sheng; Chuang, Ya-Wen

2016-01-30

Recurrent event data are commonly observed in biomedical longitudinal studies. In many instances, there exists a terminal event, which precludes the occurrence of additional repeated events, and usually there is also a nonignorable correlation between the terminal event and recurrent events. In this article, we propose a partly Aalen's additive model with a multiplicative frailty for the rate function of recurrent event process and assume a Cox frailty model for terminal event time. A shared gamma frailty is used to describe the correlation between the two types of events. Consequently, this joint model can provide the information of temporal influence of absolute covariate effects on the rate of recurrent event process, which is usually helpful in the decision-making process for physicians. An estimating equation approach is developed to estimate marginal and association parameters in the joint model. The consistency of the proposed estimator is established. Simulation studies demonstrate that the proposed approach is appropriate for practical use. We apply the proposed method to a peritonitis cohort data set for illustration. Copyright © 2015 John Wiley & Sons, Ltd.

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

PubMed

Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

2013-08-01

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage.

PubMed

Dvořáková, Zuzana; Vorlíčková, Michaela; Renčiuk, Daniel

2017-11-01

The DNA lesions, resulting from oxidative damage, were shown to destabilize human telomere four-repeat quadruplex and to alter its structure. Long telomere DNA, as a repetitive sequence, offers, however, other mechanisms of dealing with the lesion: extrusion of the damaged repeat into loop or shifting the quadruplex position by one repeat. Using circular dichroism and UV absorption spectroscopy and polyacrylamide electrophoresis, we studied consequences of lesions at different positions of the model five-repeat human telomere DNA sequences on the structure and stability of their quadruplexes in sodium and in potassium. The repeats affected by lesion are preferentially positioned as terminal overhangs of the core quadruplex structurally similar to the four-repeat one. Forced affecting of the inner repeats leads to presence of variety of more parallel folds in potassium. In sodium the designed models form mixture of two dominant antiparallel quadruplexes whose population varies with the position of the affected repeat. The shapes of quadruplex CD spectra, namely the height of dominant peaks, significantly correlate with melting temperatures. Lesion in one guanine tract of a more than four repeats long human telomere DNA sequence may cause re-positioning of its quadruplex arrangement associated with a shift of the structure to less common quadruplex conformations. The type of the quadruplex depends on the loop position and external conditions. The telomere DNA quadruplexes are quite resistant to the effect of point mutations due to the telomere DNA repetitive nature, although their structure and, consequently, function might be altered. Copyright © 2017. Published by Elsevier B.V.

A Novel de novo CDH1 Germline Variant Aids in the Classification of C-terminal E-cadherin Alterations Predicted to Escape Nonsense-Mediated mRNA Decay.

PubMed

Krempely, Kate; Karam, Rachid

2018-05-24

Most truncating CDH1 pathogenic alterations confer an elevated lifetime risk of diffuse gastric cancer and lobular breast cancer. However, transcripts containing carboxyl-terminal (C-terminal) premature stop codons have been demonstrated to escape the nonsense-mediated mRNA decay (NMD) pathway, and gastric and breast cancer risks associated with these truncations should be carefully evaluated. A female patient underwent multigene panel testing due to a personal history of invasive lobular breast cancer diagnosed at age 54, which identified the germline CDH1 nonsense alteration, c.2506G>T (p.E836*), in the last exon of the gene. Subsequent parental testing for the alteration was negative and additional short tandem repeat analysis confirmed the familial relationships and the de novo occurrence in the proband. Based on the de novo occurrence, clinical history, and rarity in general population databases, this alteration was classified as a likely pathogenic variant. This is the most C-terminal pathogenic alteration reported to date. Additionally, this alteration contributed to the classification of six other upstream CDH1 C-terminal truncating variants as pathogenic or likely pathogenic. Identifying the most distal pathogenic alteration provides evidence to classify other C-terminal truncating variants as either pathogenic or benign, a fundamental step to offering pre-symptomatic screening and prophylactic procedures to the appropriate patients. Cold Spring Harbor Laboratory Press.

Estimation of Supercapacitor Energy Storage Based on Fractional Differential Equations.

PubMed

Kopka, Ryszard

2017-12-22

In this paper, new results on using only voltage measurements on supercapacitor terminals for estimation of accumulated energy are presented. For this purpose, a study based on application of fractional-order models of supercapacitor charging/discharging circuits is undertaken. Parameter estimates of the models are then used to assess the amount of the energy accumulated in supercapacitor. The obtained results are compared with energy determined experimentally by measuring voltage and current on supercapacitor terminals. All the tests are repeated for various input signal shapes and parameters. Very high consistency between estimated and experimental results fully confirm suitability of the proposed approach and thus applicability of the fractional calculus to modelling of supercapacitor energy storage.

Quiet Short-Haul Research Airplane (QSRA) model select panel functional description

NASA Technical Reports Server (NTRS)

Watson, D. M.

1982-01-01

The QSRA, when equipped with programmable color cathode ray tube displays, a head up display, a general purpose digital computer and a microwave landing system receiver, will provide a capability to do handling qualities studies and terminal area operating systems experiments as well as to enhance an experimenter's ability to obtain repeatable aircraft performance data. The operating systems experiments include the capability to generate minimum fuel approach and departure paths and to conduct precision approaches to a STOLport runway. The mode select panel is designed to provide both the flexibility needed for a variety of flight test experiments and the minimum workload operation required by pilots flying into congested terminal traffic areas.

Repeated pregnancy among women with known HIV status in Pune, India.

PubMed

Suryavanshi, Nishi; Erande, Ashwini; Pisal, Hemlata; Shankar, Anita V; Bhosale, Ramesh A; Bollinger, Robert C; Phadke, Mrudula; Sastry, Jayagowri

2008-10-01

HIV-positive women of reproductive age face challenges in decision making related to pregnancy. Understanding factors influencing repeat pregnancies in women with known HIV status are necessary to guide interventions and counseling strategies to better inform and support them. We compared three groups of women attending a large antenatal clinic in Pune, India. They include: Group A--63 HIV-positive women coming for care for a repeat pregnancy after being diagnosed in a previous pregnancy; Group B--64 HIV-negative (repeat) pregnant women attending this antenatal clinic; and Group C--63 HIV-positive non-pregnant women currently enrolled in an ongoing clinical trial. Comparisons of Group A and B indicate that the likelihood of unplanned repeat pregnancies was significantly higher in HIV-positive (70%) than HIV-negative (36%) women (OR=4.1, CI: 2.0-8.7). Inability to terminate the pregnancy (31%) and familial obligations (40%) appear to be important for continuing the unplanned repeat pregnancy. Despite high reported contraceptive use by HIV-positive women, pregnancies still occurred. Death of their youngest child is an important factor as 21% of HIV-positive pregnant women lost their youngest child compared with 3% of HIV-negative women and 3% of HIV-positive non-pregnant women (p<0.001). Repeat pregnancies were more likely to occur for women who did not disclose their HIV status to their spouse. Thus the majority of the repeat pregnancies for HIV-positive women were both unplanned and unwanted.

[Active miniature inverted-repeat transposable elements transposon in plants: a review].

PubMed

Hu, Bingjie; Zhou, Mingbing

2018-02-25

Miniature inverted-repeat transposable elements transposon is a special transposon that could transpose by "cut-paste" mechanism, which is one of characteristics of DNA transposons. Otherwise, the copy number of MITEs is very high, which is one of characteristics of RNA transposons. Many MITE families have been reported, but little about active MITEs. We summarize recent advances in studying active MITEs. Most the MITEs belong to the Tourist-like family, such as mPing, mGing, PhTourist1, Tmi1 and PhTst-3. Additionally, DTstu1 and MITE-39 belong to Stowaway-like family, and AhMITEs1 belongs to Mutator-like family. Moreover, we summarize the structure (terminal inverse repeats and target site duplications), copy number, evolution pattern and transposition characteristics of these active MITEs, to provide the foundation for the identification of other active MITEs and subsequent research on MITE transposition and amplification mechanism.

A European mobile satellite system concept exploiting CDMA and OBP

NASA Technical Reports Server (NTRS)

Vernucci, A.; Craig, A. D.

1993-01-01

This paper describes a novel Land Mobile Satellite System (LMSS) concept applicable to networks allowing access to a large number of gateway stations ('Hubs'), utilizing low-cost Very Small Aperture Terminals (VSAT's). Efficient operation of the Forward-Link (FL) repeater can be achieved by adopting a synchronous Code Division Multiple Access (CDMA) technique, whereby inter-code interference (self-noise) is virtually eliminated by synchronizing orthogonal codes. However, with a transparent FL repeater, the requirements imposed by the highly decentralized ground segment can lead to significant efficiency losses. The adoption of a FL On-Board Processing (OBP) repeater is proposed as a means of largely recovering this efficiency impairment. The paper describes the network architecture, the system design and performance, the OBP functions and impact on implementation. The proposed concept, applicable to a future generation of the European LMSS, was developed in the context of a European Space Agency (ESA) study contract.

Insertion of reticuloendotheliosis virus long terminal repeat into the genome of CVI988 strain of Marek’s disease virus results in enhanced growth and protection

USDA-ARS?s Scientific Manuscript database

Marek’s disease (MD) is a lymphoproliferative disease of chicken caused by serotype 1 MD virus (MDV). Vaccination of commercial poultry has drastically reduced losses from MD and the poultry industry cannot be sustained without the use of vaccines. Retrovirus insertion into herpesviruses genome is a...

The Function of Neuroendocrine Cells in Prostate Cancer

DTIC Science & Technology

2013-04-01

integration site. We then performed deep sequencing and aligned reads to the genome. Our analysis revealed that both histological phenotypes are derived from...lentiviral integration site analysis . (B) Laser capture microdissection was performed on individual glands containing both squamous and...lentiviral integration site analysis . LTR: long terminal repeat (viral DNA), PCR: polymerase chain reaction. (D) Venn diagrams depict shared lentiviral

Distribution of Reading Time When Questions are Asked about a Restricted Category of Text Information. Technical Report No. 83.

ERIC Educational Resources Information Center

Reynolds, Ralph E.; And Others

Forty-three college students read a specially prepared text either with or without inserted questions. The text and the questions were presented on a computer terminal to allow measurement of reading times on short segments of material. Question groups performed better, relative to controls, on posttest items that repeated inserted questions and…

A trans-Dominant Form of Gag Restricts Ty1 Retrotransposition and Mediates Copy Number Control

PubMed Central

Saha, Agniva; Mitchell, Jessica A.; Nishida, Yuri; Hildreth, Jonathan E.; Ariberre, Joshua A.; Gilbert, Wendy V.

2015-01-01

ABSTRACT Saccharomyces cerevisiae and Saccharomyces paradoxus lack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionates with Ty1 virus-like particles (VLPs) and affects VLP yield, protein composition, and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupts sites used for VLP assembly. Glutathione S-transferase (GST) affinity pulldowns also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function and expands the strategies used to limit retroelement propagation. IMPORTANCE Retrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement, and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication is fundamentally important. The present work describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose-dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have coevolved a form of copy number control that is beneficial to both “host and parasite.” To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways in which restriction proteins modulate retroelement replication. PMID:25609815

Diversity and relationships of Crocus sativus and its relatives analysed by inter-retroelement amplified polymorphism (IRAP)

PubMed Central

Alsayied, Nouf Fakieh; Fernández, José Antonio; Schwarzacher, Trude; Heslop-Harrison, J. S.

2015-01-01

Background and Aims: Saffron (Crocus sativus) is a sterile triploid (2n = 3x = 24) cultivated species, of unknown origin from other diploid and polyploid species in the genus Crocus (Iridaceae). Species in the genus have high morphological diversity, with no clear phylogenetic patterns below the level of section Crocus series Crocus. Using DNA markers, this study aimed to examine the diversity and relationships within and between species of Crocus series Crocus. Methods: Eleven inter-retroelement amplified polymorphism (IRAP) primers were used in 63 different combinations with 35 single-plant accessions of C. sativus and related Crocus species in order to determine genetic variability and to conduct phylogenetic analysis. Key Results: A total of 4521 distinct polymorphic bands from 100 bp to approx. 4 kb were amplified; no fragment specific to all accessions of a single species was amplified. The polymorphic information content (PIC) values varied from approx. 0·37 to approx. 0·05 (mean 0·17 ± 0·1) and the major allele frequency had a mean of 0·87. High levels of polymorphism were identified between accessions of the six species of Crocus series Crocus related to C. sativus, with further variation between the species. In contrast, no polymorphisms were seen among 17 C. sativus accessions obtained in the region from Kashmir through Iran to Spain. Conclusions In contrast to the intraspecific variability seen in other Crocus species, C. sativus has minimal genetic variation, and it is concluded that the triploid hybrid species has most probably arisen only once. The data show that saffron is an allotriploid species, with the IRAP analysis indicating that the most likely ancestors are C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics, and show the need for conservation and collection of wild Crocus. PMID:26138822

Off-target effects of sulforaphane include the derepression of long terminal repeats through histone acetylation events.

PubMed

Baier, Scott R; Zbasnik, Richard; Schlegel, Vicki; Zempleni, Janos

2014-06-01

Sulforaphane is a naturally occurring isothiocyanate in cruciferous vegetables. Sulforaphane inhibits histone deacetylases, leading to the transcriptional activation of genes including tumor suppressor genes. The compound has attracted considerable attention in the chemoprevention of prostate cancer. Here we tested the hypothesis that sulforaphane is not specific for tumor suppressor genes but also activates loci such as long terminal repeats (LTRs), which might impair genome stability. Studies were conducted using chemically pure sulforaphane in primary human IMR-90 fibroblasts and in broccoli sprout feeding studies in healthy adults. Sulforaphane (2.0 μM) caused an increase in LTR transcriptional activity in cultured cells. Consumption of broccoli sprouts (34, 68 or 102 g) by human volunteers caused a dose dependent elevation in LTR mRNA in circulating leukocytes, peaking at more than a 10-fold increase. This increase in transcript levels was associated with an increase in histone H3 K9 acetylation marks in LTR 15 in peripheral blood mononuclear cells from subjects consuming sprouts. Collectively, this study suggests that sulforaphane has off-target effects that warrant further investigation when recommending high levels of sulforaphane intake, despite its promising activities in chemoprevention. Copyright © 2014 Elsevier Inc. All rights reserved.

Genome-wide Annotation and Comparative Analysis of Long Terminal Repeat Retrotransposons between Pear Species of P. bretschneideri and P. Communis

PubMed Central

Yin, Hao; Du, Jianchang; Wu, Jun; Wei, Shuwei; Xu, Yingxiu; Tao, Shutian; Wu, Juyou; Zhang, Shaoling

2015-01-01

Recent sequencing of the Oriental pear (P. bretschneideri Rehd.) genome and the availability of the draft genome sequence of Occidental pear (P. communis L.), has provided a good opportunity to characterize the abundance, distribution, timing, and evolution of long terminal repeat retrotransposons (LTR-RTs) in these two important fruit plants. Here, a total of 7247 LTR-RTs, which can be classified into 148 families, have been identified in the assembled Oriental pear genome. Unlike in other plant genomes, approximately 90% of these elements were found to be randomly distributed along the pear chromosomes. Further analysis revealed that the amplification timeframe of elements varies dramatically in different families, super-families and lineages, and the Copia-like elements have highest activity in the recent 0.5 million years (Mys). The data also showed that two genomes evolved with similar evolutionary rates after their split from the common ancestor ~0.77–1.66 million years ago (Mya). Overall, the data provided here will be a valuable resource for further investigating the impact of transposable elements on gene structure, expression, and epigenetic modification in the pear genomes. PMID:26631625

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

PubMed

De Nicola, Beatrice; Lech, Christopher J; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N; Phan, Anh Tuân

2016-07-27

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Early detection of a two-long-terminal-repeat junction molecule in the cytoplasm of recombinant murine leukemia virus-infected cells.

PubMed

Serhan, Fatima; Penaud, Magalie; Petit, Caroline; Leste-Lasserre, Thierry; Trajcevski, Stéphane; Klatzmann, David; Duisit, Ghislaine; Sonigo, Pierre; Moullier, Philippe

2004-06-01

We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19. Using cell fractionation combined with careful controls, we found that a two-long-terminal-repeat (two-LTR) junction molecule was detectable in the cytoplasm as early as 2 h post virus entry. Altogether, our data indicated that the neosynthesized retroviral DNA led to the early formation of structures including true two-LTR junctions in the cytoplasm of MLV-infected cells. Thus, the classical assumption that two-LTR circles are a mitosis-dependent dead-end product accumulating in the nucleus must be reconsidered. MLV-derived products containing a two-LTR junction can no longer be used as an exclusive surrogate for the preintegration complex nuclear translocation event.

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

PubMed Central

Thakur, Nagendra N.; El-Gogo, Susanne; Steer, Beatrix; Freimüller, Klaus; Waha, Andreas; Adler, Heiko

2007-01-01

Background Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. Methodology/Principal Findings We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. Conclusions/Significance These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression. PMID:17710133

FLG mutation p.Lys4021X in the C-terminal imperfect filaggrin repeat in Japanese patients with atopic eczema.

PubMed

Nemoto-Hasebe, I; Akiyama, M; Nomura, T; Sandilands, A; McLean, W H I; Shimizu, H

2009-12-01

Mutations in the gene encoding filaggrin (FLG) have been shown to predispose to atopic eczema (AE). Further to establish population genetics of FLG mutations in the Japanese population and to elucidate effects of FLG mutations to filaggrin biosynthesis in skin of patients with AE. We searched for FLG mutations in 19 newly recruited Japanese patients with AE. We then screened 137 Japanese patients with AE and 134 Japanese control individuals for a novel mutation identified in the present study. In addition, we evaluated FLG mRNA expression by real-time reverse transcription-polymerase chain reaction and profilaggrin/filaggrin protein expression by immunohistochemical staining in the epidermis of the patients carrying the novel mutation. We identified a novel FLG nonsense mutation c.12069A>T (p.Lys4021X) in one patient with AE. Upon further screening, p.Lys4021X was identified in four patients with AE (2.9% of all the patients with AE). In total, there are at least eight FLG variants in the Japanese population. Here we show that about 27% of patients in our Japanese AE case series carry one or more of these eight FLG mutations and these variants are also carried by 3.7% of Japanese general control individuals. There is a significant statistical association between the eight FLG mutations and AE (chi(2) P = 6.50 x 10(-8)). Interestingly, the present nonsense mutation is in the C-terminal incomplete filaggrin repeat and is the mutation nearest the C-terminal among previously reported FLG mutations. Immunohistochemical staining for filaggrin revealed that this nonsense mutation leads to remarkable reduction of filaggrin protein expression in the patients' epidermis. We clearly demonstrated that FLG mutations are significantly associated with AE in the Japanese population. The present results further support the hypothesis that the C-terminal region is essential for proper processing of profilaggrin to filaggrin.

Genome of Horsepox Virus

PubMed Central

Tulman, E. R.; Delhon, G.; Afonso, C. L.; Lu, Z.; Zsak, L.; Sandybaev, N. T.; Kerembekova, U. Z.; Zaitsev, V. L.; Kutish, G. F.; Rock, D. L.

2006-01-01

Here we present the genomic sequence of horsepox virus (HSPV) isolate MNR-76, an orthopoxvirus (OPV) isolated in 1976 from diseased Mongolian horses. The 212-kbp genome contained 7.5-kbp inverted terminal repeats and lacked extensive terminal tandem repetition. HSPV contained 236 open reading frames (ORFs) with similarity to those in other OPVs, with those in the central 100-kbp region most conserved relative to other OPVs. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of vaccinia virus (VACV) and rabbitpox virus, clearly grouping together these VACV-like viruses. Fifty-four HSPV ORFs likely represented fragments of 25 orthologous OPV genes, including in the central region the only known fragmented form of an OPV ribonucleotide reductase large subunit gene. In terminal genomic regions, HSPV lacked full-length homologues of genes variably fragmented in other VACV-like viruses but was unique in fragmentation of the homologue of VACV strain Copenhagen B6R, a gene intact in other known VACV-like viruses. Notably, HSPV contained in terminal genomic regions 17 kbp of OPV-like sequence absent in known VACV-like viruses, including fragments of genes intact in other OPVs and approximately 1.4 kb of sequence present only in cowpox virus (CPXV). HSPV also contained seven full-length genes fragmented or missing in other VACV-like viruses, including intact homologues of the CPXV strain GRI-90 D2L/I4R CrmB and D13L CD30-like tumor necrosis factor receptors, D3L/I3R and C1L ankyrin repeat proteins, B19R kelch-like protein, D7L BTB/POZ domain protein, and B22R variola virus B22R-like protein. These results indicated that HSPV contains unique genomic features likely contributing to a unique virulence/host range phenotype. They also indicated that while closely related to known VACV-like viruses, HSPV contains additional, potentially ancestral sequences absent in other VACV-like viruses. PMID:16940536

The long terminal repeat-containing retrotransposon Tf1 possesses amino acids in gag that regulate nuclear localization and particle formation.

PubMed

Kim, Min-Kyung; Claiborn, Kathryn C; Levin, Henry L

2005-08-01

Tf1 is a long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe that is studied to further our understanding of retrovirus propagation. One important application is to examine Tf1 as a model for how human immunodeficiency virus type 1 proteins enter the nucleus. The accumulation of Tf1 Gag in the nucleus requires an N-terminal nuclear localization signal (NLS) and the nuclear pore factor Nup124p. Here, we report that NLS activity is regulated by adjacent residues. Five mutant transposons were made, each with sequential tracts of four amino acids in Gag replaced by alanines. All five versions of Tf1 transposed with frequencies that were significantly lower than that of the wild type. Although all five made normal amounts of Gag, two of the mutations did not make cDNA, indicating that Gag contributed to reverse transcription. The localization of the Gag in the nucleus was significantly reduced by mutations A1, A2, and A3. These results identified residues in Gag that contribute to the function of the NLS. The Gags of A4 and A5 localized within the nucleus but exhibited severe defects in the formation of virus-like particles. Of particular interest was that the mutations in Gag-A4 and Gag-A5 caused their nuclear localization to become independent of Nup124p. These results suggested that Nup124p was only required for import of Tf1 Gag because of its extensive multimerization.

Genetic Basis for Rhizobium etli CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions▿

PubMed Central

Ojeda, Kristylea J.; Box, Jodie M.; Noel, K. Dale

2010-01-01

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis. PMID:19948805

The use of life review to enhance spiritual well-being in patients with terminal illnesses: An integrative review.

PubMed

Kwan, Cecilia W M; Ng, Marques S N; Chan, Carmen W H

2017-12-01

To conduct an integrative review of the current literature on using life review as an intervention to address the spiritual need of patients with terminal illnesses. Palliative care highlights the holistic approach of care including the spiritual aspect. Life review has been used in palliative nursing intending to enhance patients' emotional and spiritual well-being, and quality of life. However, there is a lack of publications that provide a structured overview on life review programmes and their effectiveness. Integrative review. The Whittemore and Knafl integrative review method was used. Five major online databases were included in our literature search. The keywords used were "life review" and "palliative care, terminal care, terminally ill, death & dying, hospice, spiritual wellbeing, spirituality". Seven primary papers were identified, critically appraised and synthesised in the final review. There are limited clinical studies on life review programmes for patients with terminal illness. The research design of these studies is too widely varied for meta-analysis. Here, we identified two major programmes of life review as an intervention to address the spiritual well-being of patients with terminal illness. However, repeated studies on the effectiveness of these two programmes are lacking. The shorter programme of life review is more likely to be applicable and effective for terminal patients. Further research in this area is required to provide strong evidence on the effectiveness and applicability of life review in patients receiving palliative care. This review adds weight to the need of a better understanding on the use of life review in addressing the spiritual needs of patients with terminal illness. Such understanding would provide evidence for the use of life review as an alternative approach in palliative care delivery. © 2017 John Wiley & Sons Ltd.

Insights on genome size evolution from a miniature inverted repeat transposon driving a satellite DNA.

PubMed

Scalvenzi, Thibault; Pollet, Nicolas

2014-12-01

The genome size in eukaryotes does not correlate well with the number of genes they contain. We can observe this so-called C-value paradox in amphibian species. By analyzing an amphibian genome we asked how repetitive DNA can impact genome size and architecture. We describe here our discovery of a Tc1/mariner miniature inverted-repeat transposon family present in Xenopus frogs. These transposons named miDNA4 are unique since they contain a satellite DNA motif. We found that miDNA4 measured 331 bp, contained 25 bp long inverted terminal repeat sequences and a sequence motif of 119 bp present as a unique copy or as an array of 2-47 copies. We characterized the structure, dynamics, impact and evolution of the miDNA4 family and its satellite DNA in Xenopus frog genomes. This led us to propose a model for the evolution of these two repeated sequences and how they can synergize to increase genome size. Copyright © 2014 Elsevier Inc. All rights reserved.

Origin of the polymorphism of the involucrin gene in Asians.

PubMed Central

Djian, P; Delhomme, B; Green, H

1995-01-01

The involucrin gene, encoding a protein of the terminally differentiated keratinocyte, is polymorphic in the human. There is polymorphism of marker nucleotides a two positions in the coding region, and there are over eight polymorphic forms based on the number and kind of 10-codon tandem repeats in that part of the coding region most recently added in the human lineage. The involucrin alleles of Caucasians and Africans differ in both nucleotides and repeat patterns. We show that the involucrin alleles of East Asians (Chinese and Japanese) can be divided into two populations according to whether they possess the two marker nucleotides typical of Africans or Caucasians. The Asian population bearing Caucasian-type marker nucleotides has repeat patterns similar to those of Caucasians, whereas Asians bearing African-type marker nucleotides have repeat patterns that resemble those of Africans more than those of Caucasians. The existence of two populations of East Asian involucrin alleles gives support for the existence of a Eurasian stem lineage from which Caucasians and a part of the Asian population originated. PMID:7762559

Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

PubMed

Kupiec, M; Steinlauf, R

1997-06-09

Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

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

Stochaj, Ursula; Banski, Piotr; Kodiha, Mohamed

2006-08-01

Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-{beta}1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. Inmore » ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.« less

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

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

Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier

2014-01-03

Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence uniquemore » in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.« less

Bacterial group II introns: not just splicing.

PubMed

Toro, Nicolás; Jiménez-Zurdo, José Ignacio; García-Rodríguez, Fernando Manuel

2007-04-01

Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered almost 14 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy. This review summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targetting methods. Bacterial group II intron diversity, evolutionary relationships, and behaviour in bacteria are also discussed.

DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

PubMed

Batyuk, Alexander; Wu, Yufan; Honegger, Annemarie; Heberling, Matthew M; Plückthun, Andreas

2016-04-24

DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of the DMAE-modified juvenile excretory-secretory protein Juv-p120 of Litomosoides sigmodontis.

PubMed

Wagner, Ulrike; Hirzmann, Jörg; Hintz, Martin; Beck, Ewald; Geyer, Rudolf; Hobom, Gerd; Taubert, Anja; Zahner, Horst

2011-04-01

Juv-p120 is an excretory-secretory 160 kDa glycoprotein of juvenile female Litomosoides sigmodontis and exhibits features typical for mucins. 50% of its molecular mass is attributed to posttranslational modifications with the unusual substituent dimethylaminoethanol (DMAE). By that Juv-p120 corresponds to the surface proteins of the microfilarial sheath, Shp3 and Shp3a. The secreted protein consists of 697 amino acids, organized in two different domains of repeat elements separated by a stretch of polar residues. The N-terminal domain shows fourteen P/S/T/F-rich repeat elements highly modified with phospho-DMAE substituted O-glycans confering a negative charge to the protein. The C-terminal domain is extremely rich in glutamine (35%) and leucine (25%) in less organized repeats and may play a role in oligomerization of Juv-p120 monomers. A protein family with a similar Q/L-rich region and conserved core promoter region was identified in Brugia malayi by homology screening and in Wuchereria bancrofti and Loa loa by database similarity search. One of the Q/L-rich proteins in each genus has an extended S/T-rich region and due to this feature is supposed to be a putative Juv-p120 ortholog. The corresponding modification of Juv-p120 and the microfilarial sheath surface antigens Shp3/3a explains the appearance of anti-sheath antibodies before the release of microfilariae. The function of Juv-p120 is unknown. Copyright © 2011 Elsevier B.V. All rights reserved.

Infill of tunnel valleys associated with landward-flowing ice sheets: The missing Middle Pleistocene record of the NW European rivers?

NASA Astrophysics Data System (ADS)

Moreau, Julien; Huuse, Mads

2014-01-01

The southern termination of the Middle and Late Pleistocene Scandinavian ice sheets was repeatedly located in the southern North Sea (sNS) and adjacent, north-sloping land areas. Giant meltwater-excavated valleys (tunnel valleys) formed at the southern termination of the ice sheets and contain a hitherto enigmatic succession of northward prograding clinoforms, comprising 1000s km3 of sediment. This study analyses 3D seismic data, covering the entire sNS, and demonstrates for the first time that the formation of these tunnel valleys was separate from their infill. The infill constitutes the postglacial record of the NW European river deltas, which had so far been considered missing.

DNA sequence transfer between two high-cysteine chorion gene families in the silkmoth Bombyx mori.

PubMed Central

Iatrou, K; Tsitilou, S G; Kafatos, F C

1984-01-01

We have previously shown that one type of high-cysteine silkmoth chorion protein (Hc-A) has evolved from the A family of chorion proteins by radical modifications of the NH2-terminal and COOH-terminal polypeptide arms: most of the arm sequences have been deleted, while short cysteine- and glycine-containing repeats have expanded into long arrays. Strikingly similar modifications of the arms have led to the evolution of a second type of high-cysteine protein (Hc-B) from the B family of chorion proteins. It appears that the parallel evolution of these high-cysteine-encoding gene families has not been entirely independent: examination of 3' untranslated regions shows evidence of information transfer between the two families. PMID:6589605

Identification and functional characterization of BTas transactivator as a DNA-binding protein.

PubMed

Tan, Juan; Hao, Peng; Jia, Rui; Yang, Wei; Liu, Ruichang; Wang, Jinzhong; Xi, Zhen; Geng, Yunqi; Qiao, Wentao

2010-09-30

The genome of bovine foamy virus (BFV) encodes a transcriptional transactivator, namely BTas, that remarkably enhances gene expression by binding to the viral long-terminal repeat promoter (LTR) and internal promoter (IP). In this report, we characterized the functional domains of BFV BTas. BTas contains two major functional domains: the N-terminal DNA-binding domain (residues 1-133) and the C-terminal activation domain (residues 198-249). The complete BTas responsive regions were mapped to the positions -380/-140 of LTR and 9205/9276 of IP. Four BTas responsive elements were identified at the positions -368/-346, -327/-307, -306/-285 and -186/-165 of the BFV LTR, and one element was identified at the position 9243/9264 of the BFV IP. Unlike other foamy viruses, the five BTas responsive elements in BFV shared obvious sequence homology. These data suggest that among the complex retroviruses, BFV appears to have a unique transactivation mechanism. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

Reversible ion transportation switch by a ligand-gated synthetic supramolecular ion channel.

PubMed

Muraoka, Takahiro; Endo, Takahiro; Tabata, Kazuhito V; Noji, Hiroyuki; Nagatoishi, Satoru; Tsumoto, Kouhei; Li, Rui; Kinbara, Kazushi

2014-11-05

Inspired by the regulation of cellular activities found in the ion channel proteins, here we developed membrane-embedded synthetic chiral receptors 1 and 2 with different terminal structures, where receptor 1 has hydrophobic triisopropylsilyl (TIPS) groups and receptor 2 has hydrophilic hydroxy groups. The receptors have ligand-binding units that interact with cationic amphiphiles such as 2-phenethylamine (PA). Conductance study revealed that the receptors hardly show ion transportation at the ligand-free state. After ligand binding involving a conformational change, receptor 1 bearing TIPS termini displays a significant current enhancement due to ion transportation. The current substantially diminishes upon addition of β-cyclodextrin (βCD) that scavenges the ligand from the receptor. Importantly, the receptor again turns into the conductive state by the second addition of PA, and the activation/deactivation of the ion transportation can be repeated. In contrast, receptor 2 bearing the hydroxy terminal groups hardly exhibits ion transportation, suggesting the importance of terminal TIPS groups of 1 that likely anchor the receptor in the membrane.

Olfactory CNG channel desensitization by Ca2+/CaM via the B1b subunit affects response termination but not sensitivity to recurring stimulation.

PubMed

Song, Yijun; Cygnar, Katherine D; Sagdullaev, Botir; Valley, Matthew; Hirsh, Sarah; Stephan, Aaron; Reisert, Johannes; Zhao, Haiqing

2008-05-08

Ca2+/calmodulin-mediated negative feedback is a prototypical regulatory mechanism for Ca2+-permeable ion channels. In olfactory sensory neurons (OSNs), such regulation on the cyclic nucleotide-gated (CNG) channel is considered a major mechanism of OSN adaptation. To determine the role of Ca2+/calmodulin desensitization of the olfactory CNG channel, we introduced a mutation in the channel subunit CNGB1b in mice that rendered the channel resistant to fast desensitization by Ca2+/calmodulin. Contrary to expectations, mutant OSNs showed normal receptor current adaptation to repeated stimulation. Rather, they displayed slower response termination and, consequently, reduced ability to transmit olfactory information to the olfactory bulb. They also displayed reduced response decline during sustained odorant exposure. These results suggest that Ca2+/calmodulin-mediated CNG channel fast desensitization is less important in regulating the sensitivity to recurring stimulation than previously thought and instead functions primarily to terminate OSN responses.

Scanning Tunneling Microscopy Study of Carbon Tetrachloride Adsorption and Degradation on a Natural a-Fe2O3(0001) Surface in Ultrahigh Vacuum

NASA Astrophysics Data System (ADS)

Taeg Rim, Kwang; Fitts, Jeffrey; Adib, Kaveh; Camillone, Nicholas, III; Schlosser, Peter; Osgood, Richard, Jr.; Flynn, George; Joyce, Stephen

2001-03-01

Scanning tunneling microscopy and low energy electron diffraction have been used to study a natural a-Fe2O3(0001) surface and the adsorption and degradation of carbon tetrachloride on the reduced Fe3O4(111) terminated surface. A natural a-Fe2O3 (0001) surface was prepared by repeated cycles of Ar+ ion sputtering and annealing in vacuum or in O2 at 850 K. STM images and a LEED pattern indicate that an Fe3O4(111) terminated surface and a bi-phase can be formed depending on annealing conditions. The Fe3O4(111) terminated surface was dosed with CCl4 at room temperature, and flashed up to 590 K and 850 K. STM images show adsorbates on the surface at room temperature and the degradation products of CCl4 are isolated on the surface as the flashing temperature increases up to 850 K. Results from a companion temperature programmed desorption investigation are used in conjunction with the STM images to propose site specific reactions of CCl4 on the Fe3O4(111) terminated surface.

Concerted evolution of the tandem array encoding primate U2 snRNA occurs in situ, without changing the cytological context of the RNU2 locus.

PubMed Central

Pavelitz, T; Rusché, L; Matera, A G; Scharf, J M; Weiner, A M

1995-01-01

In primates, the tandemly repeated genes encoding U2 small nuclear RNA evolve concertedly, i.e. the sequence of the U2 repeat unit is essentially homogeneous within each species but differs somewhat between species. Using chromosome painting and the NGFR gene as an outside marker, we show that the U2 tandem array (RNU2) has remained at the same chromosomal locus (equivalent to human 17q21) through multiple speciation events over > 35 million years leading to the Old World monkey and hominoid lineages. The data suggest that the U2 tandem repeat, once established in the primate lineage, contained sequence elements favoring perpetuation and concerted evolution of the array in situ, despite a pericentric inversion in chimpanzee, a reciprocal translocation in gorilla and a paracentric inversion in orang utan. Comparison of the 11 kb U2 repeat unit found in baboon and other Old World monkeys with the 6 kb U2 repeat unit in humans and other hominids revealed that an ancestral U2 repeat unit was expanded by insertion of a 5 kb retrovirus bearing 1 kb long terminal repeats (LTRs). Subsequent excision of the provirus by homologous recombination between the LTRs generated a 6 kb U2 repeat unit containing a solo LTR. Remarkably, both junctions between the human U2 tandem array and flanking chromosomal DNA at 17q21 fall within the solo LTR sequence, suggesting a role for the LTR in the origin or maintenance of the primate U2 array. Images PMID:7828589

Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes.

PubMed

De Feyter, R; Yang, Y; Gabriel, D W

1993-01-01

Six plasmid-borne avirulence (avr) genes were previously cloned from strain XcmH of the cotton pathogen, Xanthomonas campestris pv. malvacearum. We have now localized all six avr genes on the cloned fragments by subcloning and Tn5-gusA insertional mutagenesis. None of these avr genes appeared to exhibit exclusively gene-for-gene patterns of interactions with cotton R genes, and avrB4 was demonstrated to confer avr gene-for-R genes (plural) avirulence to X. c. pv. malvacearum on congenic cotton lines carrying either of two different resistance loci, B1 or B4. Furthermore, the B1 locus appeared to confer R gene-for-avr genes resistance to cotton against isogenic X. c. pv. malvacearum strains carrying any one of three avr genes: avrB4, avrb6, or avrB102. Restriction enzyme, Southern blot hybridization, and DNA sequence analyses showed that the XcmH avr genes are all highly similar to each other, to avrBs3 and avrBsP from the pepper pathogen X. c. pv. vesicatoria, and to the host-specific virulence gene pthA from the citrus pathogen X. citri. The XcmH avr genes differed primarily in the multiplicity of a tandemly repeated 102-base pair motif within the central portions of the genes, repeated from 14 to 23 times in members of this gene family. The complete nucleotide sequence of avrb6 revealed that it is 97% identical in DNA sequence to avrB4, avrBs3, avrBsP, and pthA and that 62-bp inverted terminal repeats mark the boundaries of homology between avrb6 and all members of this Xanthomonas virulence/avirulence gene family sequenced to date. The terminal 38 bp of both inverted repeats are highly similar to the 38-bp consensus terminal sequence of the Tn3 family of transposons. Up to 11 members of the avr gene family appear to be present in North American strains of X. c. pv. malvacearum, including XcmH. The high level of homology observed among these avr genes and their presence in multiple copies may explain the gene-for-genes interactions and also the observed high frequencies (10(-3) to 10(-4) per locus) of X. c. pv. malvacearum race change mutations. Five spontaneous race change mutants of XcmH suffered avr locus deletions, strongly indicating intergenic recombination as the primary mechanism for generating new races in X. c. pv. malvacearum.

From NGS assembly challenges to instability of fungal mitochondrial genomes: A case study in genome complexity.

PubMed

Misas, Elizabeth; Muñoz, José Fernando; Gallo, Juan Esteban; McEwen, Juan Guillermo; Clay, Oliver Keatinge

2016-04-01

The presence of repetitive or non-unique DNA persisting over sizable regions of a eukaryotic genome can hinder the genome's successful de novo assembly from short reads: ambiguities in assigning genome locations to the non-unique subsequences can result in premature termination of contigs and thus overfragmented assemblies. Fungal mitochondrial (mtDNA) genomes are compact (typically less than 100 kb), yet often contain short non-unique sequences that can be shown to impede their successful de novo assembly in silico. Such repeats can also confuse processes in the cell in vivo. A well-studied example is ectopic (out-of-register, illegitimate) recombination associated with repeat pairs, which can lead to deletion of functionally important genes that are located between the repeats. Repeats that remain conserved over micro- or macroevolutionary timescales despite such risks may indicate functionally or structurally (e.g., for replication) important regions. This principle could form the basis of a mining strategy for accelerating discovery of function in genome sequences. We present here our screening of a sample of 11 fully sequenced fungal mitochondrial genomes by observing where exact k-mer repeats occurred several times; initial analyses motivated us to focus on 17-mers occurring more than three times. Based on the diverse repeats we observe, we propose that such screening may serve as an efficient expedient for gaining a rapid but representative first insight into the repeat landscapes of sparsely characterized mitochondrial chromosomes. Our matching of the flagged repeats to previously reported regions of interest supports the idea that systems of persisting, non-trivial repeats in genomes can often highlight features meriting further attention. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins.

PubMed

Sun, D; Leung, C L; Liem, R K

2001-01-01

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3' end of the MACF cDNA; hence, we refer to it as MACF2. The C-terminal domain of mouse MACF2 was cloned and characterized. The microtubule-binding properties of MACF2 C-terminal domain are similar to that of MACF. The GAR domain was originally found in Gas 2 protein and here we show that it can associate with MTs in transfected cells. Plectin and desmoplakin have GSR-containing domains at their C-termini and we further demonstrate that the GSR-containing domain of plectin, but not desmoplakin, can bind to MTs in vivo.

The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres

PubMed Central

Marzullo, Marta; Raffa, Grazia D.; Morciano, Patrizia; Raimondo, Domenico; Burla, Romina; Saggio, Isabella; Gatti, Maurizio

2015-01-01

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping. PMID:26110638

Analysis of a kinetic multi-segment foot model. Part I: Model repeatability and kinematic validity.

PubMed

Bruening, Dustin A; Cooney, Kevin M; Buczek, Frank L

2012-04-01

Kinematic multi-segment foot models are still evolving, but have seen increased use in clinical and research settings. The addition of kinetics may increase knowledge of foot and ankle function as well as influence multi-segment foot model evolution; however, previous kinetic models are too complex for clinical use. In this study we present a three-segment kinetic foot model and thorough evaluation of model performance during normal gait. In this first of two companion papers, model reference frames and joint centers are analyzed for repeatability, joint translations are measured, segment rigidity characterized, and sample joint angles presented. Within-tester and between-tester repeatability were first assessed using 10 healthy pediatric participants, while kinematic parameters were subsequently measured on 17 additional healthy pediatric participants. Repeatability errors were generally low for all sagittal plane measures as well as transverse plane Hindfoot and Forefoot segments (median<3°), while the least repeatable orientations were the Hindfoot coronal plane and Hallux transverse plane. Joint translations were generally less than 2mm in any one direction, while segment rigidity analysis suggested rigid body behavior for the Shank and Hindfoot, with the Forefoot violating the rigid body assumptions in terminal stance/pre-swing. Joint excursions were consistent with previously published studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Efficient production of artificially designed gelatins with a Bacillus brevis system.

PubMed

Kajino, T; Takahashi, H; Hirai, M; Yamada, Y

2000-01-01

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Basidiomycete fungal communities in Australian sclerophyll forest soil are altered by repeated prescribed burning.

PubMed

Anderson, Ian C; Bastias, Brigitte A; Genney, David R; Parkin, Pamela I; Cairney, John W G

2007-04-01

Soil basidiomycetes play key roles in forest nutrient and carbon cycling processes, yet the diversity and structure of below ground basidiomycete communities remain poorly understood. Prescribed burning is a commonly used forest management practice and there is evidence that single fire events can have an impact on soil fungal communities but little is known about the effects of repeated prescribed burning. We have used internal transcribed spacer (ITS) terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the impacts of repeated prescribed burning every two or four years over a period of 30 years on soil basidiomycete communities in an Australian wet sclerophyll forest. Detrended correspondence analysis of ITS T-RFLP profiles separated basidiomycete communities in unburned control plots from those in burned plots, with those burned every two years being the most different from controls. Burning had no effect on basidiomycete species richness, thus these differences appear to be due to changes in community structure. Basidiomycete communities in the unburned control plots were vertically stratified in the upper 20 cm of soil, but no evidence was found for stratification in the burned plots, suggesting that repeated prescribed burning results in more uniform basidiomycete communities. Overall, the results demonstrate that repeated prescribed burning alters soil basidiomycete communities, with the effect being greater with more frequent burning.

The Ups and Downs of Repeated Cleavage and Internal Fragment Production in Top-Down Proteomics.

PubMed

Lyon, Yana A; Riggs, Dylan; Fornelli, Luca; Compton, Philip D; Julian, Ryan R

2018-01-01

Analysis of whole proteins by mass spectrometry, or top-down proteomics, has several advantages over methods relying on proteolysis. For example, proteoforms can be unambiguously identified and examined. However, from a gas-phase ion-chemistry perspective, proteins are enormous molecules that present novel challenges relative to peptide analysis. Herein, the statistics of cleaving the peptide backbone multiple times are examined to evaluate the inherent propensity for generating internal versus terminal ions. The raw statistics reveal an inherent bias favoring production of terminal ions, which holds true regardless of protein size. Importantly, even if the full suite of internal ions is generated by statistical dissociation, terminal ions are predicted to account for at least 50% of the total ion current, regardless of protein size, if there are three backbone dissociations or fewer. Top-down analysis should therefore be a viable approach for examining proteins of significant size. Comparison of the purely statistical analysis with actual top-down data derived from ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD) reveals that terminal ions account for much of the total ion current in both experiments. Terminal ion production is more favored in UVPD relative to HCD, which is likely due to differences in the mechanisms controlling fragmentation. Importantly, internal ions are not found to dominate from either the theoretical or experimental point of view. Graphical abstract ᅟ.

Prenatal detection of a de novo terminal inverted duplication 4p in a fetus with the Wolf-Hirschhorn syndrome phenotype.

PubMed

Beaujard, M-P; Jouannic, J-M; Bessières, B; Borie, C; Martin-Luis, I; Fallet-Bianco, C; Portnoï, M-F

2005-06-01

To present the prenatal diagnosis of a de novo terminal inversion duplication of the short arm of chromosome 4 and a review of the literature. An amniocentesis for chromosome analysis was performed at 33 weeks' gestation because ultrasound examination showed a female fetus with multiple abnormalities consisting of severe intrauterine growth retardation, microcephaly, a cleft lip and renal hypoplasia. Cytogenetic analysis and FISH studies of the cultured amniocytes revealed a de novo terminal inversion duplication of the short arm of chromosome 4 characterized by a duplication of 4p14-p16.1 chromosome region concomitant with a terminal deletion 4p16.1-pter. The karyotype was thus: 46,XX, inv dup del (4)(:p14-->p16.1::p16.1-->qter). The parents opted to terminate the pregnancy. Fetopathological examination showed dysmorphic features and abnormalities consistent with a Wolf-Hirschhorn syndrome (WHS) diagnosis, clinical manifestations of partial 4p trisomy being mild. Although relatively rare, inverted duplications have been reported repeatedly in an increasing number of chromosomes. Only two previous cases with de novo inv dup del (4p) and one with tandem dup 4p have been reported, all of them associated with a 4pter deletion. We report the first case diagnosed prenatally. Breakpoints are variable, resulting in different abnormal phenotype. In our case, clinical manifestations resulted in a WHS phenotype.

The Ups and Downs of Repeated Cleavage and Internal Fragment Production in Top-Down Proteomics

NASA Astrophysics Data System (ADS)

Lyon, Yana A.; Riggs, Dylan; Fornelli, Luca; Compton, Philip D.; Julian, Ryan R.

2018-01-01

Analysis of whole proteins by mass spectrometry, or top-down proteomics, has several advantages over methods relying on proteolysis. For example, proteoforms can be unambiguously identified and examined. However, from a gas-phase ion-chemistry perspective, proteins are enormous molecules that present novel challenges relative to peptide analysis. Herein, the statistics of cleaving the peptide backbone multiple times are examined to evaluate the inherent propensity for generating internal versus terminal ions. The raw statistics reveal an inherent bias favoring production of terminal ions, which holds true regardless of protein size. Importantly, even if the full suite of internal ions is generated by statistical dissociation, terminal ions are predicted to account for at least 50% of the total ion current, regardless of protein size, if there are three backbone dissociations or fewer. Top-down analysis should therefore be a viable approach for examining proteins of significant size. Comparison of the purely statistical analysis with actual top-down data derived from ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD) reveals that terminal ions account for much of the total ion current in both experiments. Terminal ion production is more favored in UVPD relative to HCD, which is likely due to differences in the mechanisms controlling fragmentation. Importantly, internal ions are not found to dominate from either the theoretical or experimental point of view. [Figure not available: see fulltext.

Identification and Characterization of Multiple Spidroin 1 Genes Encoding Major Ampullate Silk Proteins in Nephila clavipes

PubMed Central

Gaines, William A.; Marcotte, William R.

2010-01-01

Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSp) that consist of a large repeat array flanked by non-repetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We have experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species. PMID:18828837

Mitochondrial genome of the tomato clownfish Amphiprion frenatus (Pomacentridae, Amphiprioninae).

PubMed

Ye, Le; Hu, Jing; Wu, Kaichang; Wang, Yu; Li, Jianlong

2016-01-01

The complete mitochondrial (mt) genome of the tomato clownfish Amphiprion frenatus was obtained in this study. The circular mtDNA molecule was 16,774 bp in size and the overall nucleotide composition of the H-strand was 29.72% A, 25.81% T, 15.38% G and 29.09% C, with an A + T bias. The complete mitogenome encoded 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region (D-loop), with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. The D-loop included termination associated sequence (TAS), central conserved domain (CCD) and conserved sequence block (CSB), and was composed of 6 complete continuity tandem repeat units and an imperfect tandem repeat unit.

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization.

PubMed

Allan, Rudi K; Mok, Danny; Ward, Bryan K; Ratajczak, Thomas

2006-03-17

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed

Blochlinger, K; Diggelmann, H

1984-12-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed Central

Blochlinger, K; Diggelmann, H

1984-01-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells. Images PMID:6098829

Cabling design for phased arrays

NASA Technical Reports Server (NTRS)

Kruger, I. D.; Turkiewicz, L.

1972-01-01

The ribbon-cabling system used for the AEGIS phased array which provides minimum cable bulk, complete EMI shielding, rugged mechanical design, repeatable electrical characteristics, and ease of assembly and maintenance is described. The ribbon cables are 0.040-inch thick, and in widths up to 2 1/2 inches. Their terminations are molded connectors that can be grouped in a three-tier arrangement, with cable branching accomplished by a matrix-welding technique.

Synaptic behaviors of thin-film transistor with a Pt/HfO x /n-type indium-gallium-zinc oxide gate stack.

PubMed

Yang, Paul; Park, Daehoon; Beom, Keonwon; Kim, Hyung Jun; Kang, Chi Jung; Yoon, Tae-Sik

2018-07-20

We report a variety of synaptic behaviors in a thin-film transistor (TFT) with a metal-oxide-semiconductor gate stack that has a Pt/HfO x /n-type indium-gallium-zinc oxide (n-IGZO) structure. The three-terminal synaptic TFT exhibits a tunable synaptic weight with a drain current modulation upon repeated application of gate and drain voltages. The synaptic weight modulation is analog, voltage-polarity dependent reversible, and strong with a dynamic range of multiple orders of magnitude (>10 4 ). This modulation process emulates biological synaptic potentiation, depression, excitatory-postsynaptic current, paired-pulse facilitation, and short-term to long-term memory transition behaviors as a result of repeated pulsing with respect to the pulse amplitude, width, repetition number, and the interval between pulses. These synaptic behaviors are interpreted based on the changes in the capacitance of the Pt/HfO x /n-IGZO gate stack, the channel mobility, and the threshold voltage that result from the redistribution of oxygen ions by the applied gate voltage. These results demonstrate the potential of this structure for three-terminal synaptic transistor using the gate stack composed of the HfO x gate insulator and the IGZO channel layer.

Long Terminal Repeat Retrotransposon Content in Eight Diploid Sunflower Species Inferred from Next-Generation Sequence Data

PubMed Central

Tetreault, Hannah M.; Ungerer, Mark C.

2016-01-01

The most abundant transposable elements (TEs) in plant genomes are Class I long terminal repeat (LTR) retrotransposons represented by superfamilies gypsy and copia. Amplification of these superfamilies directly impacts genome structure and contributes to differential patterns of genome size evolution among plant lineages. Utilizing short-read Illumina data and sequence information from a panel of Helianthus annuus (sunflower) full-length gypsy and copia elements, we explore the contribution of these sequences to genome size variation among eight diploid Helianthus species and an outgroup taxon, Phoebanthus tenuifolius. We also explore transcriptional dynamics of these elements in both leaf and bud tissue via RT-PCR. We demonstrate that most LTR retrotransposon sublineages (i.e., families) display patterns of similar genomic abundance across species. A small number of LTR retrotransposon sublineages exhibit lineage-specific amplification, particularly in the genomes of species with larger estimated nuclear DNA content. RT-PCR assays reveal that some LTR retrotransposon sublineages are transcriptionally active across all species and tissue types, whereas others display species-specific and tissue-specific expression. The species with the largest estimated genome size, H. agrestis, has experienced amplification of LTR retrotransposon sublineages, some of which have proliferated independently in other lineages in the Helianthus phylogeny. PMID:27233667

Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains.

PubMed

Hayes, Michael L; Giang, Karolyn; Berhane, Beniam; Mulligan, R Michael

2013-12-20

Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins are required as RNA binding specificity determinants in the RNA editing mechanism. Bioinformatic analysis has shown that most of the Arabidopsis PPR proteins necessary for RNA editing events include a C-terminal portion that shares structural characteristics with a superfamily of deaminases. The DYW deaminase domain includes a highly conserved zinc binding motif that shares characteristics with cytidine deaminases. The Arabidopsis PPR genes, ELI1 and DOT4, both have DYW deaminase domains and are required for single RNA editing events in chloroplasts. The ELI1 DYW deaminase domain was expressed as a recombinant protein in Escherichia coli and was shown to bind two zinc atoms per polypeptide. Thus, the DYW deaminase domain binds a zinc metal ion, as expected for a cytidine deaminase, and is potentially the catalytic component of an editing complex. Genetic complementation experiments demonstrate that large portions of the DYW deaminase domain of ELI1 may be eliminated, but the truncated genes retain the ability to restore editing site conversion in a mutant plant. These results suggest that the catalytic activity can be supplied in trans by uncharacterized protein(s) of the editosome.

The coactivator CBP stimulates human T-cell lymphotrophic virus type I Tax transactivation in vitro.

PubMed

Kashanchi, F; Duvall, J F; Kwok, R P; Lundblad, J R; Goodman, R H; Brady, J N

1998-12-18

Tax interacts with the cellular cyclic AMP-responsive element binding protein (CREB) and facilitates the binding of the coactivator CREB binding protein (CBP), forming a multimeric complex on the cyclic AMP-responsive element (CRE)-like sites in the human T-cell lymphotrophic virus type I (HTLV-I) promoter. The trimeric complex is believed to recruit additional regulatory proteins to the HTLV-I long terminal repeat, but there has been no direct evidence that CBP is required for Tax-mediated transactivation. We present evidence that Tax and CBP activate transcription from the HTLV-I 21 base pair repeats on naked DNA templates. Transcriptional activation of the HTLV-I sequences required both Tax and CBP and could be mediated by either the N-terminal activation domain of CBP or the full-length protein. Fluorescence polarization binding assays indicated that CBP does not markedly enhance the affinity of Tax for the trimeric complex. Transcription analyses suggest that CBP activates Tax-dependent transcription by promoting transcriptional initiation and reinitiation. The ability of CBP to activate the HTLV-I promoter does not involve the stabilization of Tax binding, but rather depends upon gene activation properties of the co-activator that function in the context of a naked DNA template.

Coactivator-associated arginine methyltransferase 1 enhances transcriptional activity of the human T-cell lymphotropic virus type 1 long terminal repeat through direct interaction with Tax.

PubMed

Jeong, Soo-Jin; Lu, Hanxin; Cho, Won-Kyung; Park, Hyeon Ung; Pise-Masison, Cynthia; Brady, John N

2006-10-01

In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

Synaptic behaviors of thin-film transistor with a Pt/HfO x /n-type indium–gallium–zinc oxide gate stack

NASA Astrophysics Data System (ADS)

Yang, Paul; Park, Daehoon; Beom, Keonwon; Kim, Hyung Jun; Kang, Chi Jung; Yoon, Tae-Sik

2018-07-01

We report a variety of synaptic behaviors in a thin-film transistor (TFT) with a metal-oxide-semiconductor gate stack that has a Pt/HfO x /n-type indium–gallium–zinc oxide (n-IGZO) structure. The three-terminal synaptic TFT exhibits a tunable synaptic weight with a drain current modulation upon repeated application of gate and drain voltages. The synaptic weight modulation is analog, voltage-polarity dependent reversible, and strong with a dynamic range of multiple orders of magnitude (>104). This modulation process emulates biological synaptic potentiation, depression, excitatory-postsynaptic current, paired-pulse facilitation, and short-term to long-term memory transition behaviors as a result of repeated pulsing with respect to the pulse amplitude, width, repetition number, and the interval between pulses. These synaptic behaviors are interpreted based on the changes in the capacitance of the Pt/HfO x /n-IGZO gate stack, the channel mobility, and the threshold voltage that result from the redistribution of oxygen ions by the applied gate voltage. These results demonstrate the potential of this structure for three-terminal synaptic transistor using the gate stack composed of the HfO x gate insulator and the IGZO channel layer.

The Long Terminal Repeat Retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe.

PubMed

Esnault, Caroline; Levin, Henry L

2015-08-01

The long terminal repeat (LTR) retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe are active mobile elements of the Ty3/gypsy family. The mobilization of these retrotransposons depends on particle formation, reverse transcription and integration, processes typical of other LTR retrotransposons. However, Tf1 and Tf2 are distinct from other LTR elements in that they assemble virus-like particles from a single primary translation product, initiate reverse transcription with an unusual self-priming mechanism, and, in the case of Tf1, integrate with a pattern that favors specific promoters of RNA pol II-transcribed genes. To avoid the chromosome instability and genome damage that results from increased copy number, S. pombe applies a variety of defense mechanisms that restrict Tf1 and Tf2 activity. The mRNA of the Tf elements is eliminated by an exosome-based pathway when cells are in favorable conditions whereas nutrient deprivation triggers an RNA interference-dependent pathway that results in the heterochromatization of the elements. Interestingly, Tf1 integrates into the promoters of stress-induced genes and these insertions are capable of increasing the expression of adjacent genes. These properties of Tf1 transposition raise the possibility that Tf1 benefits cells with specific insertions by providing resistance to environmental stress.

An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression.

PubMed

Kiermer, V; Van Lint, C; Briclet, D; Vanhulle, C; Kettmann, R; Verdin, E; Burny, A; Droogmans, L

1998-07-01

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

Non-terminal blood sampling techniques in guinea pigs.

PubMed

Birck, Malene M; Tveden-Nyborg, Pernille; Lindblad, Maiken M; Lykkesfeldt, Jens

2014-10-11

Guinea pigs possess several biological similarities to humans and are validated experimental animal models(1-3). However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages(4,5). Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals(6). All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox.

PubMed

Gubser, Caroline; Smith, Geoffrey L

2002-04-01

Camelpox virus (CMPV) and variola virus (VAR) are orthopoxviruses (OPVs) that share several biological features and cause high mortality and morbidity in their single host species. The sequence of a virulent CMPV strain was determined; it is 202182 bp long, with inverted terminal repeats (ITRs) of 6045 bp and has 206 predicted open reading frames (ORFs). As for other poxviruses, the genes are tightly packed with little non-coding sequence. Most genes within 25 kb of each terminus are transcribed outwards towards the terminus, whereas genes within the centre of the genome are transcribed from either DNA strand. The central region of the genome contains genes that are highly conserved in other OPVs and 87 of these are conserved in all sequenced chordopoxviruses. In contrast, genes towards either terminus are more variable and encode proteins involved in host range, virulence or immunomodulation. In some cases, these are broken versions of genes found in other OPVs. The relationship of CMPV to other OPVs was analysed by comparisons of DNA and predicted protein sequences, repeats within the ITRs and arrangement of ORFs within the terminal regions. Each comparison gave the same conclusion: CMPV is the closest known virus to variola virus, the cause of smallpox.

TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization

PubMed Central

Galati, Alessandra; Micheli, Emanuela; Alicata, Claudia; Ingegnere, Tiziano; Cicconi, Alessandro; Pusch, Miriam Caroline; Giraud-Panis, Marie-Josèphe; Gilson, Eric; Cacchione, Stefano

2015-01-01

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection. PMID:25999344

TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein*

PubMed Central

Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

2012-01-01

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes. PMID:22696218

Immune Responses Induced by Gene Gun or Intramuscular Injection of DNA Vaccines That Express Immunogenic Regions of the Serine Repeat Antigen from Plasmodium falciparum

PubMed Central

Belperron, Alexia A.; Feltquate, David; Fox, Barbara A.; Horii, Toshihiro; Bzik, David J.

1999-01-01

The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein. PMID:10496891

Identification of a Novel Virulence Determinant with Serum Opacification Activity in Streptococcus suis

PubMed Central

Baums, Christoph G.; Kaim, Ute; Fulde, Marcus; Ramachandran, Girish; Goethe, Ralph; Valentin-Weigand, Peter

2006-01-01

Streptococcus suis serotype 2 is a porcine and human pathogen with adhesive and invasive properties. In other streptococci, large surface-associated proteins (>100 kDa) of the MSCRAMM family (microbial surface components recognizing adhesive matrix molecules) are key players in interactions with host tissue. In this study, we identified a novel opacity factor of S. suis (OFS) with structural homology to members of the MSCRAMM family. The N-terminal region of OFS is homologous to the respective regions of fibronectin-binding protein A (FnBA) of Streptococcus dysgalactiae and the serum opacity factor (SOF) of Streptococcus pyogenes. Similar to these two proteins, the N-terminal domain of OFS opacified horse serum. Serum opacification activity was detectable in sodium dodecyl sulfate extracts of wild-type S. suis but not in extracts of isogenic ofs knockout mutants. Heterologous expression of OFS in Lactococcus lactis demonstrated that a high level of expression of OFS is sufficient to provide surface-associated serum opacification activity. Furthermore, serum opacification could be inhibited by an antiserum against recombinant OFS. The C-terminal repetitive sequence elements of OFS differed significantly from the respective repeat regions of FnBA and SOF as well as from the consensus sequence of the fibronectin-binding repeats of MSCRAMMs. Accordingly, fibronectin binding was not detectable in recombinant OFS. To investigate the putative function of OFS in the pathogenesis of invasive S. suis diseases, piglets were experimentally infected with an isogenic mutant strain in which the ofs gene had been knocked out by an in-frame deletion. The mutant was severely attenuated in virulence but not in colonization, demonstrating that OFS represents a novel virulence determinant of S. suis. PMID:17057090

Two divergent Symbiodinium genomes reveal conservation of a gene cluster for sunscreen biosynthesis and recently lost genes.

PubMed

Shoguchi, Eiichi; Beedessee, Girish; Tada, Ipputa; Hisata, Kanako; Kawashima, Takeshi; Takeuchi, Takeshi; Arakaki, Nana; Fujie, Manabu; Koyanagi, Ryo; Roy, Michael C; Kawachi, Masanobu; Hidaka, Michio; Satoh, Noriyuki; Shinzato, Chuya

2018-06-14

The marine dinoflagellate, Symbiodinium, is a well-known photosynthetic partner for coral and other diverse, non-photosynthetic hosts in subtropical and tropical shallows, where it comprises an essential component of marine ecosystems. Using molecular phylogenetics, the genus Symbiodinium has been classified into nine major clades, A-I, and one of the reported differences among phenotypes is their capacity to synthesize mycosporine-like amino acids (MAAs), which absorb UV radiation. However, the genetic basis for this difference in synthetic capacity is unknown. To understand genetics underlying Symbiodinium diversity, we report two draft genomes, one from clade A, presumed to have been the earliest branching clade, and the other from clade C, in the terminal branch. The nuclear genome of Symbiodinium clade A (SymA) has more gene families than that of clade C, with larger numbers of organelle-related genes, including mitochondrial transcription terminal factor (mTERF) and Rubisco. While clade C (SymC) has fewer gene families, it displays specific expansions of repeat domain-containing genes, such as leucine-rich repeats (LRRs) and retrovirus-related dUTPases. Interestingly, the SymA genome encodes a gene cluster for MAA biosynthesis, potentially transferred from an endosymbiotic red alga (probably of bacterial origin), while SymC has completely lost these genes. Our analysis demonstrates that SymC appears to have evolved by losing gene families, such as the MAA biosynthesis gene cluster. In contrast to the conservation of genes related to photosynthetic ability, the terminal clade has suffered more gene family losses than other clades, suggesting a possible adaptation to symbiosis. Overall, this study implies that Symbiodinium ecology drives acquisition and loss of gene families.

Carboxy-terminal sequence variation of LMP1 gene in Epstein-Barr-virus-associated mononucleosis and tumors from Serbian patients.

PubMed

Banko, Ana; Lazarevic, Ivana; Cupic, Maja; Stevanovic, Goran; Boricic, Ivan; Jovanovic, Tanja

2012-04-01

Seven strains of Epstein-Barr virus (EBV) are defined based on C-terminal sequence variations of the latent membrane protein 1 (LMP1). Some strains, especially those with a 30-bp deletion, are thought to be related to tumorigenic activity and geographical localization. The aims of the study were to determine the prevalence of different LMP1 strains and to investigate sequence variation in the C-terminal region of LMP1 in Serbian isolates. This study included 53 EBV-DNA-positive plasma and tissue block samples from patients with mononucleosis syndrome, renal transplantation, and tumors, mostly nasopharyngeal carcinoma. The sequence of the 506-bp fragment of LMP1 C terminus was used for phylogenetic analyses and identification of LMP1 strains, deletions, and mutations. The majority of isolates were non-deleted (66%), and the rest had 30-bp, rare 69-bp, or yet unknown 27-bp deletions, which were not related to malignant or non-malignant isolate origin. However, the majority of 69-bp deletion isolates were derived from patients with nasopharyngeal carcinoma. Less than five 33-bp repeats were found in the majority of non-deleted isolates (68.6%), whereas most 69-bp deletion isolates (75%) had five or six repeats. Serbian isolates were assigned to four LMP1 strains: B95-8 (32.1%), China 1 (24.5%), North Carolina (NC; 18.9%), and Mediterranean (Med; 24.5%). In NC isolates, three new mutations unique for this strain were identified. EBV EBNA2 genotypes 1 and 2 were both found, with dominance of genotype 1 (90.7%). This study demonstrated noticeable geographical-associated characteristics in the LMP1 C terminus of investigated isolates. Copyright © 2012 Wiley Periodicals, Inc.

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

PubMed

Itoh, Aiko; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

2017-11-01

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Repeated Carbon-Cycle Disturbances at the Permian-Triassic Boundary Separate two Mass Extinctions

NASA Astrophysics Data System (ADS)

Nicol, J. A.; Watson, L.; Claire, M.; Buick, R.; Catling, D. C.

2004-12-01

Non-marine organic matter in Permian-Triassic sediments from the Blue Mountains, eastern Australia shows seven negative δ13C excursions of up to 7%, terminating with a positive excursion of 4%. Fluctuations start at the late Permian Glossopteris floral extinction and continue until just above the palynological Permian-Triassic boundary, correlated with the peak of marine mass extinction. The isotopic fluctuations are not linked to changes in depositional setting, kerogen composition or plant community, so they evidently resulted from global perturbations in atmospheric δ13C and/or CO2. The pattern was not produced by a single catastrophe such as a meteorite impact, and carbon-cycle calculations indicate that gas release during flood-basalt volcanism was insufficient. Methane-hydrate melting can generate a single -7% shift, but cannot produce rapid multiple excursions without repeated reservoir regeneration and release. However, the data are consistent with repeated overturning of a stratified ocean, expelling toxic gases that promoted sequential mass extinctions in the terrestrial and marine realms.

Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum.

PubMed

Robinson, Graham C; Vegunta, Yogesh; Gabus, Caroline; Gaubitz, Christl; Thore, Stéphane

2017-05-01

The Target of Rapamycin Complex is a central controller of cell growth and differentiation in eukaryotes. Its global architecture has been described by cryoelectron microscopy, and regions of its central TOR protein have been described by X-ray crystallography. However, the N-terminal region of this protein, which consists of a series of HEAT repeats, remains uncharacterised at high resolution, most likely due to the absence of a suitable purification procedure. Here, we present a robust method for the preparation of the HEAT-repeat domain, utilizing the thermophilic fungus Chaetomium thermophilum as a source organism. We describe construct design and stable expression in insect cells. An efficient two-step purification procedure is presented, and the purified product is characterised by SEC and MALDI-TOF MS. The methods described pave the way for a complete high-resolution characterisation of this elusive region of the TOR protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Duodenoscope reprocessing surveillance with adenosine triphosphate testing and terminal cultures: a clinical pilot study.

PubMed

Visrodia, Kavel; Hanada, Yuri; Pennington, Kelly M; Tosh, Pritish K; Topazian, Mark D; Petersen, Bret T

2017-07-01

Recent reports of infectious outbreaks linked to duodenoscopes have led to proposals for duodenoscope surveillance culturing, which has inherent limitations. We aimed to assess the feasibility of real-time adenosine triphosphate (ATP) testing after manual cleaning and its ability to predict reprocessing adequacy, as determined by terminal duodenoscope cultures. Clinically used duodenoscopes underwent reprocessing per current guidelines. After manual cleaning, ATP samples were obtained from the elevator, within the proximal biopsy port, and by flushing of the biopsy channel. After high-level disinfection (HLD), aerobic cultures of the elevator and biopsy channel were obtained using sterile technique. Duodenoscopes with any ATP sample ≥200 relative light units underwent repeated cycles of cleaning, ATP testing, HLD, and terminal culturing. Twenty clinically used duodenoscopes were included; 18 underwent a second reprocessing cycle, and 6 underwent a third reprocessing cycle because of detection of high ATP. After the initial reprocessing cycle, 12 of 20 (60%) duodenoscopes had positive culture results, most commonly yielding gram-negative bacilli (GNB, n = 11 from 9 duodenoscopes), and catalase-positive gram-positive cocci (CP-GPC, n = 7 from 7 duodenoscopes), suggesting staphylococcal organisms. Ambient environmental controls also showed GNB and CP-GPC growth. The overall sensitivity and specificity of ATP testing compared with terminal cultures were 30% and 53%, respectively. ATP sampling appears to correlate poorly with terminal culture results and cannot be recommended as a surrogate for terminal cultures. The performance and interpretation of cultures remains complicated by the potential recovery of environmental contaminants. Copyright © 2017 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

PubMed

Yarimizu, Tohru; Nakamura, Mikiko; Hoshida, Hisashi; Akada, Rinji

2015-02-14

Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences. Mutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield. We present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

5.625 Gbps bidirectional laser communications measurements between the NFIRE satellite and an optical ground station

NASA Astrophysics Data System (ADS)

Fields, Renny A.; Kozlowski, David A.; Yura, Harold T.; Wong, Robert L.; Wicker, Josef M.; Lunde, Carl T.; Gregory, Mark; Wandernoth, Bernhard K.; Heine, Frank F.; Luna, Joseph J.

2011-11-01

5.625 Gbps bidirectional laser communication at 1064 nm has been demonstrated on a repeatable basis between a Tesat coherent laser communication terminal with a 6.5 cm diameter ground aperture mounted inside the European Space Agency Optical Ground Station dome at Izana, Tenerife and a similar space-based terminal (12.4 cm diameter aperture) on the Near-Field InfraRed Experiment (NFIRE) low-earth-orbiting spacecraft. Both night and day bidirectional links were demonstrated with the longest being 177 seconds in duration. Correlation with atmospheric models and preliminary atmospheric r0 and scintillation measurements have been made for the conditions tested, suggesting that such coherent systems can be deployed successfully at still lower altitudes without resorting to the use of adaptive optics for compensation.

Low-Temperature Carrier Transport in Ionic-Liquid-Gated Hydrogen-Terminated Silicon

NASA Astrophysics Data System (ADS)

Sasama, Yosuke; Yamaguchi, Takahide; Tanaka, Masashi; Takeya, Hiroyuki; Takano, Yoshihiko

2017-11-01

We fabricated ionic-liquid-gated field-effect transistors on the hydrogen-terminated (111)-oriented surface of undoped silicon. Ion implantation underneath electrodes leads to good ohmic contacts, which persist at low temperatures down to 1.4 K. The sheet resistance of the channel decreases by more than five orders of magnitude as the gate voltage is changed from 0 to -1.6 V at 220 K. This is caused by the accumulation of hole carriers. The sheet resistance shows thermally activated behavior at temperatures below 10 K, which is attributed to hopping transport of the carriers. The activation energy decreases towards zero with increasing carrier density, suggesting the approach to an insulator-metal transition. We also report the variation of device characteristics induced by repeated sweeps of the gate voltage.

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.

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

PubMed Central

Khan, A S; Repaske, R; Garon, C F; Chan, H W; Rowe, W P; Martin, M A

1982-01-01

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA. Images PMID:6281459

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1×1 Nucleotide UU Internal Loop Conformations⊥

PubMed Central

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W.; Disney, Matthew D.

2011-01-01

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5′CUG/3′GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures are disclosed of a model DM1 triplet repeating construct, 5′r(UUGGGC(CUG)3GUCC)2, refined to 2.20 Å and 1.52 Å resolution. Here, differences in orientation of the 5′ dangling UU end between the two structures induce changes in the backbone groove width, which reveals that non-canonical 1×1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5′UU forms one hydrogen-bonded pairs with a 5′UU of a neighboring helix in the unit cell to form a pseudo-infinite helix. The central 1×1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1×1 nucleotide UU internal loops each form a one hydrogen-bonded pair. In the 1.52 Å structure, CUGb, the 5′ UU dangling end is tucked into the major groove of the duplex. While the canonical paired bases show no change in base pairing, in CUGb the terminal 1×1 nucleotide UU internal loops form now two hydrogen-bonded pairs. Thus, the shift in major groove induced by the 5′UU dangling end alters non-canonical base patterns. Collectively, these structures indicate that 1×1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands. PMID:21988728

Myotonic dystrophy type 1 RNA crystal structures reveal heterogeneous 1 × 1 nucleotide UU internal loop conformations.

PubMed

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W; Disney, Matthew D

2011-11-15

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[UUGGGC(CUG)(3)GUCC](2), refined to 2.20 and 1.52 Å resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 × 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 × 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 × 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 Å structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 × 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 × 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

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

Kumar, Amit; Park, HaJeung; Fang, Pengfei

2012-03-27

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[{und UU}GGGC(C{und U}G){sub 3}GUCC]{sub 2}, refined to 2.20 and 1.52 {angstrom} resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 x 1 nucleotide UU internal loops can display an ensemble of pairing conformations.more » In the 2.20 {angstrom} structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 x 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 x 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 {angstrom} structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 x 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 x 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.« less

Rad59-Facilitated Acquisition of Y′ Elements by Short Telomeres Delays the Onset of Senescence

PubMed Central

Churikov, Dmitri; Charifi, Ferose; Simon, Marie-Noëlle; Géli, Vincent

2014-01-01

Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1–3-like tracts present between subtelomeric X and Y′ elements, which is followed by BIR-mediated non-reciprocal translocation of Y′ element and terminal TG1–3 repeats from the donor end onto the shortened telomere. We found that choice of the Y′ donor was not random, since both engineered telomere VII-L and native VI-R acquired Y′ elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y′ translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1–3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y′ translocation events taking place during presenescence are genetically separable from Rad51-dependent Y′ amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y′ translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation. PMID:25375789

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

PubMed

Draeger, Christian; Ndinyanka Fabrice, Tohnyui; Gineau, Emilie; Mouille, Grégory; Kuhn, Benjamin M; Moller, Isabel; Abdou, Marie-Therese; Frey, Beat; Pauly, Markus; Bacic, Antony; Ringli, Christoph

2015-06-24

Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

Novel regulation of Skp1 by the Dictyostelium AgtA α-galactosyltransferase involves the Skp1-binding activity of its WD40 repeat domain.

PubMed

Schafer, Christopher M; Sheikh, M Osman; Zhang, Dongmei; West, Christopher M

2014-03-28

The role of Skp1 as an adaptor protein that links Cullin-1 to F-box proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is well characterized. In the social amoeba Dictyostelium and probably many other unicellular eukaryotes, Skp1 is modified by a pentasaccharide attached to a hydroxyproline near its C terminus. This modification is important for oxygen-sensing during Dictyostelium development and is mediated by a HIF-α type prolyl 4-hydroxylase and five sequentially acting cytoplasmic glycosyltransferase activities. Gene disruption studies show that AgtA, the enzyme responsible for addition of the final two galactose residues, in α-linkages to the Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent terminal development. AgtA possesses a WD40 repeat domain C-terminal to its single catalytic domain and, by use of domain deletions, binding studies, and enzyme assays, we find that the WD40 repeats confer a salt-sensitive second-site binding interaction with Skp1 that mediates novel catalytic activation in addition to simple substrate recognition. In addition, AgtA binds similarly well to precursor isoforms of Skp1 by a salt-sensitive mechanism that competes with binding to an F-box protein and recognition by early modification enzymes, and the effect of binding is diminished when AgtA modifies Skp1. Genetic studies show that loss of AgtA is more severe when an earlier glycosylation step is blocked, and overexpressed AgtA is deleterious if catalytically inactivated. Together, the findings suggest that AgtA mediates non-enzymatic control of unmodified and substrate precursor forms of Skp1 by a binding mechanism that is normally relieved by switch-like activation of its glycosylation function.

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

PubMed Central

Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

2005-01-01

Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

PubMed

Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

2003-08-01

Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.

Multiple functions of the leucine-rich repeat protein LrrA of Treponema denticola.

PubMed

Ikegami, Akihiko; Honma, Kiyonobu; Sharma, Ashu; Kuramitsu, Howard K

2004-08-01

The gene lrrA, encoding a leucine-rich repeat protein, LrrA, that contains eight consensus tandem repeats of 23 amino acid residues, has been identified in Treponema denticola ATCC 35405. A leucine-rich repeat is a generally useful protein-binding motif, and proteins containing this repeat are typically involved in protein-protein interactions. Southern blot analysis demonstrated that T. denticola ATCC 35405 expresses the lrrA gene, but the gene was not identified in T. denticola ATCC 33520. In order to analyze the functions of LrrA in T. denticola, an lrrA-inactivated mutant of strain ATCC 35405 and an lrrA gene expression transformant of strain ATCC 33520 were constructed. Characterization of the mutant and transformant demonstrated that LrrA is associated with the extracytoplasmic fraction of T. denticola and expresses multifunctional properties. It was demonstrated that the attachment of strain ATCC 35405 to HEp-2 cell cultures and coaggregation with Tannerella forsythensis were attenuated by the lrrA mutation. In addition, an in vitro binding assay demonstrated specific binding of LrrA to a portion of the Tannerella forsythensis leucine-rich repeat protein, BspA, which is mediated by the N-terminal region of LrrA. It was also observed that the lrrA mutation caused a reduction of swarming in T. denticola ATCC 35405 and consequently attenuated tissue penetration. These results suggest that the leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells and coaggregation with Tannerella forsythensis. These properties may play important roles in the virulence of T. denticola.

Awareness in contextual cueing of visual search as measured with concurrent access- and phenomenal-consciousness tasks.

PubMed

Schlagbauer, Bernhard; Müller, Hermann J; Zehetleitner, Michael; Geyer, Thomas

2012-10-25

In visual search, context information can serve as a cue to guide attention to the target location. When observers repeatedly encounter displays with identical target-distractor arrangements, reaction times (RTs) are faster for repeated relative to nonrepeated displays, the latter containing novel configurations. This effect has been termed "contextual cueing." The present study asked whether information about the target location in repeated displays is "explicit" (or "conscious") in nature. To examine this issue, observers performed a test session (after an initial training phase in which RTs to repeated and nonrepeated displays were measured) in which the search stimuli were presented briefly and terminated by visual masks; following this, observers had to make a target localization response (with accuracy as the dependent measure) and indicate their visual experience and confidence associated with the localization response. The data were examined at the level of individual displays, i.e., in terms of whether or not a repeated display actually produced contextual cueing. The results were that (a) contextual cueing was driven by only a very small number of about four actually learned configurations; (b) localization accuracy was increased for learned relative to nonrepeated displays; and (c) both consciousness measures were enhanced for learned compared to nonrepeated displays. It is concluded that contextual cueing is driven by only a few repeated displays and the ability to locate the target in these displays is associated with increased visual experience.

Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin.

PubMed

Papanikolopoulou, Katerina; Forge, Vincent; Goeltz, Pierrette; Mitraki, Anna

2004-03-05

The folding of beta-structured, fibrous proteins is a largely unexplored area. A class of such proteins is used by viruses as adhesins, and recent studies revealed novel beta-structured motifs for them. We have been studying the folding and assembly of adenovirus fibers that consist of a globular C-terminal domain, a central fibrous shaft, and an N-terminal part that attaches to the viral capsid. The globular C-terminal, or "head" domain, has been postulated to be necessary for the trimerization of the fiber and might act as a registration signal that directs its correct folding and assembly. In this work, we replaced the head of the fiber by the trimerization domain of the bacteriophage T4 fibritin, termed "foldon." Two chimeric proteins, comprising the foldon domain connected at the C-terminal end of four fiber shaft repeats with or without the use of a natural linker sequence, fold into highly stable, SDS-resistant trimers. The structural signatures of the chimeric proteins as seen by CD and infrared spectroscopy are reported. The results suggest that the foldon domain can successfully replace the fiber head domain in ensuring correct trimerization of the shaft sequences. Biological implications and implications for engineering highly stable, beta-structured nanorods are discussed.

Interactive computer graphics system for structural sizing and analysis of aircraft structures

NASA Technical Reports Server (NTRS)

Bendavid, D.; Pipano, A.; Raibstein, A.; Somekh, E.

1975-01-01

A computerized system for preliminary sizing and analysis of aircraft wing and fuselage structures was described. The system is based upon repeated application of analytical program modules, which are interactively interfaced and sequence-controlled during the iterative design process with the aid of design-oriented graphics software modules. The entire process is initiated and controlled via low-cost interactive graphics terminals driven by a remote computer in a time-sharing mode.

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

Complete Genome Sequence of a Naturally Occurring Simian Foamy Virus Isolate from Rhesus Macaque (SFVmmu_K3T).

PubMed

Nandakumar, Subhiksha; Bae, Eunhae H; Khan, Arifa S

2017-08-17

The full-length genome sequence of a simian foamy virus (SFVmmu_K3T), isolated from a rhesus macaque ( Macaca mulatta ), was obtained using high-throughput sequencing. SFVmmu_K3T consisted of 12,983 bp and had a genomic organization similar to that of other SFVs, with long terminal repeats (LTRs) and open reading frames for Gag, Pol, Env, Tas, and Bet.

The Processes Involved in Designing Software.

DTIC Science & Technology

1980-08-01

repeats Itself at the next level, terminating with a plan whose individual steps can be executed to solve the Initial problem. Hayes-Roth and Hayes-Roth...that the original design problem is decomposed into a collection of well structured subproblems under the control of some type of executive process...given element to refine further, the schema is assumed to execute to completion, developing a solution model for that element and refining it into a

Diversity and structure of PIF/Harbinger-like elements in the genome of Medicago truncatula

PubMed Central

Grzebelus, Dariusz; Lasota, Slawomir; Gambin, Tomasz; Kucherov, Gregory; Gambin, Anna

2007-01-01

Background Transposable elements constitute a significant fraction of plant genomes. The PIF/Harbinger superfamily includes DNA transposons (class II elements) carrying terminal inverted repeats and producing a 3 bp target site duplication upon insertion. The presence of an ORF coding for the DDE/DDD transposase, required for transposition, is characteristic for the autonomous PIF/Harbinger-like elements. Based on the above features, PIF/Harbinger-like elements were identified in several plant genomes and divided into several evolutionary lineages. Availability of a significant portion of Medicago truncatula genomic sequence allowed for mining PIF/Harbinger-like elements, starting from a single previously described element MtMaster. Results Twenty two putative autonomous, i.e. carrying an ORF coding for TPase and complete terminal inverted repeats, and 67 non-autonomous PIF/Harbinger-like elements were found in the genome of M. truncatula. They were divided into five families, MtPH-A5, MtPH-A6, MtPH-D,MtPH-E, and MtPH-M, corresponding to three previously identified and two new lineages. The largest families, MtPH-A6 and MtPH-M were further divided into four and three subfamilies, respectively. Non-autonomous elements were usually direct deletion derivatives of the putative autonomous element, however other types of rearrangements, including inversions and nested insertions were also observed. An interesting structural characteristic – the presence of 60 bp tandem repeats – was observed in a group of elements of subfamily MtPH-A6-4. Some families could be related to miniature inverted repeat elements (MITEs). The presence of empty loci (RESites), paralogous to those flanking the identified transposable elements, both autonomous and non-autonomous, as well as the presence of transposon insertion related size polymorphisms, confirmed that some of the mined elements were capable for transposition. Conclusion The population of PIF/Harbinger-like elements in the genome of M. truncatula is diverse. A detailed intra-family comparison of the elements' structure proved that they proliferated in the genome generally following the model of abortive gap repair. However, the presence of tandem repeats facilitated more pronounced rearrangements of the element internal regions. The insertion polymorphism of the MtPH elements and related MITE families in different populations of M. truncatula, if further confirmed experimentally, could be used as a source of molecular markers complementary to other marker systems. PMID:17996080

High Fat Diet Augments Amphetamine Sensitization in Mice: Role of Feeding Pattern, Obesity, and Dopamine Terminal Changes

PubMed Central

Fordahl, Steve C.; Locke, Jason L.; Jones, Sara R.

2016-01-01

High fat (HF) diet-induced obesity has been shown to augment behavioral responses to psychostimulants that target the dopamine system. The purpose of this study was to characterize dopamine terminal changes induced by a HF diet that correspond with enhanced locomotor sensitization to amphetamine. C57BL/6J mice had limited (2hr 3d/week) or extended (24h 7d/week) access to a HF diet or standard chow for six weeks. Mice were then repeatedly exposed to amphetamine (AMPH), and their locomotor responses to an amphetamine challenge were measured. Fast scan cyclic voltammetry was used to identify changes in dopamine terminal function after AMPH exposure. Exposure to a HF diet reduced dopamine uptake and increased locomotor responses to acute, high-dose AMPH administration compared to chow fed mice. Microdialysis showed elevated extracellular dopamine in the nucleus accumbens (NAc) coincided with enhanced locomotion after acute AMPH in HF-fed mice. All mice exhibited locomotor sensitization to amphetamine, but both extended and limited access to a HF diet augmented this response. Neither HF-fed group showed the robust amphetamine sensitization-induced increases in dopamine release, reuptake, and amphetamine potency observed in chow fed animals. However, the potency of amphetamine as an uptake inhibitor was significantly elevated after sensitization in mice with extended (but not limited) access to HF. Conversely, after amphetamine sensitization, mice with limited (but not extended) access to HF displayed reduced autoreceptor sensitivity to the D2/D3 agonist quinpirole. Additionally, we observed reduced membrane dopamine transporter (DAT) levels after HF, and a shift in DAT localization to the cytosol was detected with limited access to HF. This study showed that different patterns of HF exposure produced distinct dopamine terminal adaptations to repeated AMPH, which differed from chow fed mice, and enhanced sensitization to AMPH. Locomotor sensitization in chow fed mice coincided with elevated DAT function and increased AMPH potency; however, the enhanced behavioral response to AMPH after HF exposure was unique in that it coincided with reduced DAT function and diet pattern-specific adaptations. PMID:27267686

The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences

PubMed Central

2010-01-01

Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is a feasible goal. PMID:20609256

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

PubMed Central

Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

2015-01-01

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

PubMed

Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

2015-12-17

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast.

PubMed

Zhao, Xiaohong; Park, Yang-Nim; Todor, Horia; Moomau, Christine; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

2012-07-06

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

Improved material-bagging device

DOEpatents

Wach, C.G.; Nelson, R.E.; Brak, S.B.

1982-01-19

A bagging device for transferring material; specifically contaminated material, from one chamber through an opening in a wall to a second chamber includes a cylindrical housing communicating with the opening and defining a passage between the chambers. A cylindrical cartridge is slidably received within the housing. The cartridge has a substantially rigid cylindrical sleeve to which is affixed a pliable tube. The pliable tube is positioned concentrically about the sleeve and has a pleated portion capable of unfolding from the sleeve and a closed end extending over a terminal end of the sleeve. Sealing means are interposed in sealed relationship between the cartridge and the housing. Material from one chamber is inserted into the cartridge secured in the housing and received in the closed end of the tube which unfolds into the other chamber enclosing the material therein. The tube may then be sealed behind the material and then severed to form a bag-like enclosure defined by the tube's closed terminal end and the new seal. The new seal then forms a terminal end for the unsevered portion of the pliable tube into which additional material may be placed and the bagging process repeated.

Cargo selection by specific kinesin light chain 1 isoforms.

PubMed

Woźniak, Marcin J; Allan, Victoria J

2006-11-29

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.

Cargo selection by specific kinesin light chain 1 isoforms

PubMed Central

Woźniak, Marcin J; Allan, Victoria J

2006-01-01

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures. PMID:17093494

Material bagging device

DOEpatents

Wach, Charles G.; Nelson, Robert E.; Brak, Stephen B.

1984-01-01

A bagging device for transferring material from one chamber through an opening in a wall to a second chamber includes a cylindrical housing communicating with the opening and defining a passage between the chambers. A cylindrical cartridge is slidably received within the housing. The cartridge has a substantially rigid cylindrical sleeve to which is affixed a pliable tube. The pliable tube is positioned concentrically about the sleeve and has a pleated portion capable of unfolding from the sleeve and a closed end extending over a terminal end of the sleeve. Sealing means are interposed in sealed relationship between the cartridge and the housing. Material from one chamber is inserted into the cartridge secured in the housing and received in the closed end of the tube which unfolds into the other chamber enclosing the material therein. The tube may then be sealed behind the material and then severed to form a bag-like enclosure defined by the tube's closed terminal end and the new seal. The new seal then forms a terminal end for the unsevered portion of the pliable tube into which additional material may be placed and the bagging process repeated.

A TPR domain–containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B

PubMed Central

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H.J.; Snel, Berend

2013-01-01

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B–dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization. PMID:23569217

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

PubMed

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

2013-04-15

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Reversal of a Neurospora Translocation by Crossing over Involving Displaced Rdna, and Methylation of the Rdna Segments That Result from Recombination

PubMed Central

Perkins, David D.; Metzenberg, Robert L.; Raju, Namboori B.; Selker, Eric U.; Barry, Edward G.

1986-01-01

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation x Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered. PMID:2947829

The 28S–18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor

PubMed Central

Schnare, Murray N.; Collings, James C.; Spencer, David F.; Gray, Michael W.

2000-01-01

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from ∼11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an ∼55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A′ pre-rRNA processing sites within the 5′ external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5′ ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C.fasciculata and Trypanosoma brucei involves 3′-terminal addition of three A residues that are not present in the corresponding DNA sequences. PMID:10982863

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

PubMed

Tasaki, E; Hirayama, J; Tazumi, A; Hayashi, K; Hara, Y; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

2012-02-01

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

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

Koonin, Eugene V., E-mail: koonin@ncbi.nlm.nih.gov; Dolja, Valerian V., E-mail: doljav@science.oregonstate.edu; Krupovic, Mart, E-mail: krupovic@pasteur.fr

2015-05-15

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangiblemore » clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources along with additional acquisitions of diverse genes. - Highlights: • Eukaryotic virome dramatically differs from the viromes of bacteria and archaea. • Eukaryotic virome is dominated by RNA viruses and retroelements. • All classes of eukaryotic viruses evolved by gene module exchange. • Prokaryotic ancestry is traceable for core gene modules of most eukaryotic viruses. • Evolutionary histories of viruses and transposable elements are tightly linked.« less

The biological function of antibodies induced by the RTS,S/AS01 malaria vaccine candidate is determined by their fine specificity.

PubMed

Chaudhury, Sidhartha; Ockenhouse, Christian F; Regules, Jason A; Dutta, Sheetij; Wallqvist, Anders; Jongert, Erik; Waters, Norman C; Lemiale, Franck; Bergmann-Leitner, Elke

2016-05-31

Recent vaccine studies have shown that the magnitude of an antibody response is often insufficient to explain efficacy, suggesting that characteristics regarding the quality of the antibody response, such as its fine specificity and functional activity, may play a major role in protection. Previous studies of the lead malaria vaccine candidate, RTS,S, have shown that circumsporozoite protein (CSP)-specific antibodies and CD4(+) T cell responses are associated with protection, however the role of fine specificity and biological function of CSP-specific antibodies remains to be elucidated. Here, the relationship between fine specificity, opsonization-dependent phagocytic activity and protection in RTS,S-induced antibodies is explored. A new method for measuring the phagocytic activity mediated by CSP-specific antibodies in THP-1 cells is presented and applied to samples from a recently completed phase 2 RTS,S/AS01 clinical trial. The fine specificity of the antibody response was assessed using ELISA against three antigen constructs of CSP: the central repeat region, the C-terminal domain and the full-length protein. A multi-parameter analysis of phagocytic activity and fine-specificity data was carried out to identify potential correlates of protection in RTS,S. Results from the newly developed assay revealed that serum samples from RTS,S recipients displayed a wide range of robust and repeatable phagocytic activity. Phagocytic activity was correlated with full-length CSP and C-terminal specific antibody titres, but not to repeat region antibody titres, suggesting that phagocytic activity is primarily driven by C-terminal antibodies. Although no significant difference in overall phagocytic activity was observed with respect to protection, phagocytic activity expressed as 'opsonization index', a relative measure that normalizes phagocytic activity with CS antibody titres, was found to be significantly lower in protected subjects than non-protected subjects. Opsonization index was identified as a surrogate marker of protection induced by the RTS,S/AS01 vaccine and determined how antibody fine specificity is linked to opsonization activity. These findings suggest that the role of opsonization in protection in the RTS,S vaccine may be more complex than previously thought, and demonstrate how integrating multiple immune measures can provide insight into underlying mechanisms of immunity and protection.

Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

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

Tan, Kemin; Cao, Nan; Cheng, Bokun

The DNA topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug-resistant tuberculosis. In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetracysteine motifs seen in the C-terminal domains of Escherichia coli topoisomerase I. A construct (amino acids A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) andmore » the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze single-stranded DNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52 angstrom resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel beta-sheet stabilized by a crossing-over alpha-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria.« less

Running wheel exercise before a binge regimen of methamphetamine does not protect against striatal dopaminergic damage.

PubMed

O'dell, Steven J; Marshall, John F

2014-09-01

Repeated administration of methamphetamine (mAMPH) to rodents in a single-day "binge" dosing regimen produces long-lasting damage to forebrain dopaminergic nerve terminals as measured by decreases in tissue dopamine (DA) content and levels of the plasmalemmal DA transporter (DAT). However, the midbrain cell bodies from which the DA terminals arise survive, and previous reports show that striatal DA markers return to control levels by 12 months post-mAMPH, suggesting long-term repair or regrowth of damaged DA terminals. We previously showed that when rats engaged in voluntary aerobic exercise for 3 weeks before and 3 weeks after a binge regimen of mAMPH, exercise significantly ameliorated mAMPH-induced decreases in striatal DAT. However, these data left unresolved the question of whether exercise protected against the initial neurotoxicity from the mAMPH binge or accelerated the repair of the damaged DA terminals. The present experiments were designed to test whether exercise protects against the mAMPH-induced injury. Adult male Sprague-Dawley rats were allowed to run in wheels for 3 weeks before an acute binge regimen of mAMPH or saline, then placed into nonwheel cages for an additional week before autoradiographic determination of striatal DAT binding. The autoradiographic findings showed that prior exercise provided no protection against mAMPH-induced damage to striatal DA terminals. These results, together with analyses from our previous experiments, suggest that voluntary exercise may accelerate the repair of mAMPH-damaged DA terminals and that voluntary exercise may be useful as therapeutic adjunct in the treatment mAMPH addicts. © 2014 Wiley Periodicals, Inc.

Behavioral Context of Call Production by Eastern North Pacific Blue Whales

DTIC Science & Technology

2007-01-25

pairs occurring in a repeated song sequence; B calls from a different blue whale are also evident; spectrogram parameters: fast Fourier transform (FFT...Acoustic data were viewed in spectrogram form ( fast Fourier transform [FFT] length 1 s, 80% overlap, Hanning window) to de- termine the presence of calls...dura- tion to song A and B units (Table 2), but the intermit - tent timing clearly distinguishes them from song. Whales producing singular calls were

Prognostic Significance of Loss-of-Heterozygosity of the CUTL1 Putative Tumor Suppressor Gene in Breast Cancers

DTIC Science & Technology

2000-08-01

the mouse mammary tumor virus long terminal repeat (MMTV-LTR) frequently develop mammary tumors and uterine leiomyomas (4). The results of...coimmunoprecipitation analyses revealed that specific complexes of CDP/Cut and PyV LT antigen could be detected in both leiomyomas and mammary tumors (4...Martinsoudant, N., Nepveu, A., Cardiff, R. D., and Muller, W. J. The Induction Of Uterine Leiomyomas and Mammary Tumors In Transgenic Mice Expressing

Standard Engineering Installation Package, Technical Control Facility, Technical Control Improvement Program (TCIP)

DTIC Science & Technology

1979-10-01

software. This document may not be cited for purpose of advertisement . AVAILABILITY This publication is available to non-Government agencies and may be...voltage interface requirements for repeaters. This supply occupies one slot in the 10306 mounting shelf. 1.4.2.8.5 Teletypewriter monitor. The AN/ UGC -61X...The AN/ UGC -61X keyboard send-receive (KSR) TTY set is an electromechanical apparatus that provides terminal facilities with two receive only, one KSR

Influence of sex on performance fatigability of the plantar flexors following repeated maximal dynamic shortening contractions.

PubMed

Lanning, Amelia C; Power, Geoffrey A; Christie, Anita D; Dalton, Brian H

2017-10-01

The purpose was to determine sex differences in fatigability during maximal, unconstrained velocity, shortening plantar flexions. The role of time-dependent measures (i.e., rate of torque development, rate of velocity development, and rate of neuromuscular activation) in such sex-related differences was also examined. By task termination, females exhibited smaller reductions in power and similar changes in rate of neuromuscular activation than males, indicating females were less fatigable than males.

Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage.

PubMed

Kawasaki, Takeru; Shimizu, Mio; Satsuma, Hideki; Fujiwara, Akiko; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

2009-01-01

PhiRSB1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The phiRSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of phiRSB1 were demonstrated.

Large-scale modelling of the divergent spectrin repeats in nesprins: giant modular proteins.

PubMed

Autore, Flavia; Pfuhl, Mark; Quan, Xueping; Williams, Aisling; Roberts, Roland G; Shanahan, Catherine M; Fraternali, Franca

2013-01-01

Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.

Characterization of transposable elements in the ectomycorrhizal fungus Laccaria bicolor.

PubMed

Labbé, Jessy; Murat, Claude; Morin, Emmanuelle; Tuskan, Gerald A; Le Tacon, François; Martin, Francis

2012-01-01

The publicly available Laccaria bicolor genome sequence has provided a considerable genomic resource allowing systematic identification of transposable elements (TEs) in this symbiotic ectomycorrhizal fungus. Using a TE-specific annotation pipeline we have characterized and analyzed TEs in the L. bicolor S238N-H82 genome. TEs occupy 24% of the 60 Mb L. bicolor genome and represent 25,787 full-length and partial copy elements distributed within 171 families. The most abundant elements were the Copia-like. TEs are not randomly distributed across the genome, but are tightly nested or clustered. The majority of TEs exhibits signs of ancient transposition except some intact copies of terminal inverted repeats (TIRS), long terminal repeats (LTRs) and a large retrotransposon derivative (LARD) element. There were three main periods of TE expansion in L. bicolor: the first from 57 to 10 Mya, the second from 5 to 1 Mya and the most recent from 0.5 Mya ago until now. LTR retrotransposons are closely related to retrotransposons found in another basidiomycete, Coprinopsis cinerea. This analysis 1) represents an initial characterization of TEs in the L. bicolor genome, 2) contributes to improve genome annotation and a greater understanding of the role TEs played in genome organization and evolution and 3) provides a valuable resource for future research on the genome evolution within the Laccaria genus.

Role of C-terminal residues in oligomerization and stability of lambda CII: implications for lysis-lysogeny decision of the phage.

PubMed

Datta, Ajit Bikram; Roy, Siddhartha; Parrack, Pradeep

2005-01-14

A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions. Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different. The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed.

Tax relieves transcriptional repression by promoting histone deacetylase 1 release from the human T-cell leukemia virus type 1 long terminal repeat.

PubMed

Lu, Hanxin; Pise-Masison, Cynthia A; Linton, Rebecca; Park, Hyeon Ung; Schiltz, R Louis; Sartorelli, Vittorio; Brady, John N

2004-07-01

Expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. In this study, we have analyzed the role of histone deacetylase 1 (HDAC1) on HTLV-1 gene expression from an integrated template. First we show that trichostatin A, an HDAC inhibitor, enhances Tax expression in HTLV-1-transformed cells. Second, using a cell line containing a single-copy HTLV-1 long terminal repeat, we demonstrate that overexpression of HDAC1 represses Tax transactivation. Furthermore, a chromatin immunoprecipitation assay allowed us to analyze the interaction of transcription factors, coactivators, and HDACs with the basal and activated HTLV-1 promoter. We demonstrate that HDAC1 is associated with the inactive, but not the Tax-transactivated, HTLV-1 promoter. In vitro and in vivo glutathione S-transferase-Tax pull-down and coimmunoprecipitation experiments demonstrated that there is a direct physical association between Tax and HDAC1. Importantly, biotinylated chromatin pull-down assays demonstrated that Tax inhibits and/or dissociates the binding of HDAC1 to the HTLV-1 promoter. Our results provide evidence that Tax interacts directly with HDAC1 and regulates binding of the repressor to the HTLV-1 promoter.

Discontinuation of dialysis with eculizumab therapy in a pediatric patient with dense deposit disease.

PubMed

Tran, Cheryl L; Sethi, Sanjeev; Murray, David; Cramer, Carl H; Sas, David J; Willrich, Maria; Smith, Richard J; Fervenza, Fernando C

2016-04-01

Dense deposit disease (DDD) is a rare glomerular disease caused by an uncontrolled activation of the alternative complement pathway leading to end-stage renal disease in 50 % of patients. As such, DDD has been classified within the spectrum of complement component 3 (C3) glomerulopathies due to its pathogenesis from alternative pathway dysregulation. Conventional immunosuppressive therapies have no proven effectiveness. Eculizumab, a terminal complement inhibitor, has been reported to mitigate disease in some cases. We report on the efficacy of eculizumab in a pediatric patient who failed to respond to cyclophosphamide, corticosteroids, and plasma exchange. Complement biomarker profiling was remarkable for low serum C3, low properdin, and elevated soluble C5b-9. Consistent with these findings, the alternative pathway functional assay was abnormally low, indicative of alternative pathway activity, although neither C3-nephritic factors nor Factor H autoantibodies were detected. Eculizumab therapy was associated with significant improvement in proteinuria and renal function allowing discontinuation of hemodialysis (HD). Repeat C3 and soluble C5b-9 levels normalized, showing that terminal complement pathway activity was successfully blocked while the patient was receiving eculizumab therapy. Repeat testing for alternative pathway activation allowed for a successful decrease in eculizumab dosing. The case reported here demonstrates the successful recovery of renal function in a pediatric patient on HD following the use of eculizumab.

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

PubMed Central

Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

1993-01-01

A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

PubMed Central

Ryden, T A; de Mars, M; Beemon, K

1993-01-01

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive. Images PMID:8386280

Characterization of Transposable Elements in the Ectomycorrhizal Fungus Laccaria bicolor

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

Labbe, Jessy L; Murat, Claude; Morin, Emmanuelle

2012-01-01

Background: The publicly available Laccaria bicolor genome sequence has provided a considerable genomic resource allowing systematic identification of transposable elements (TEs) in this symbiotic ectomycorrhizal fungus. Using a TEspecific annotation pipeline we have characterized and analyzed TEs in the L. bicolor S238N-H82 genome. Methodology/Principal Findings: TEs occupy 24% of the 60 Mb L. bicolor genome and represent 25,787 full-length and partial copy elements distributed within 171 families. The most abundant elements were the Copia-like. TEs are not randomly distributed across the genome, but are tightly nested or clustered. The majority of TEs exhibits signs of ancient transposition except some intactmore » copies of terminal inverted repeats (TIRS), long terminal repeats (LTRs) and a large retrotransposon derivative (LARD) element. There were three main periods of TE expansion in L. bicolor: the first from 57 to 10 Mya, the second from 5 to 1 Mya and the most recent from 0.5 Mya ago until now. LTR retrotransposons are closely related to retrotransposons found in another basidiomycete, Coprinopsis cinerea. Conclusions: This analysis 1) represents an initial characterization of TEs in the L. bicolor genome, 2) contributes to improve genome annotation and a greater understanding of the role TEs played in genome organization and evolution and 3) provides a valuable resource for future research on the genome evolution within the Laccaria genus.« less

Characterization of Transposable Elements in Laccaria bicolor

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

Labbe, Jessy L; Murat, Claude; Morin, Emmanuelle

2012-01-01

Background: The publicly available Laccaria bicolor genome sequence has provided a considerable genomic resource allowing systematic identification of transposable elements (TEs) in this symbiotic ectomycorrhizal fungus. Using a TE-specific annotation pipeline we have characterized and analyzed TEs in the L. bicolor S238N-H82 genome. Methodology/Principal Findings: TEs occupy 24% of the 60 Mb L. bicolor genome and represent 25,787 full-length and partial copies elements distributed within 172 families. The most abundant elements were the Copia-like. TEs are not randomly distributed across the genome, but are tightly nested or clustered. The majority of TEs are ancient except some terminal inverted repeats (TIRS),more » long terminal repeats (LTRs) and a large retrotransposon derivative (LARD) element. There were three main periods of TEs expansion in L. bicolor; the first from 57 to 10 Mya, the second from 5 to 1 Mya and the most recent from 500,000 years ago until now. LTR retrotransposons are closely related to retrotransposons found in another basidiomycete, Coprinopsis cinerea. Conclusions: This analysis represents an initial characterization of TEs in the L. bicolor genome, contributes to genome assembly and to a greater understanding of the role TEs played in genome organization and evolution, and provides a valuable resource for the ongoing Laccaria Pan-Genome project supported by the U.S.-DOE Joint Genome Institute.« less

Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

PubMed Central

Haas, Kalina T; Lee, MiYoung; Esposito, Alessandro; Venkitaraman, Ashok R

2018-01-01

Abstract RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30–40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut—but do not overlap—with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2. PMID:29309696

Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush).

PubMed

Zhuo, L; Reed, K M; Phillips, R B

1995-06-01

Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.

Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors

PubMed Central

Wan, Hua; Hu, Jian-ping; Li, Kang-shun; Tian, Xu-hong; Chang, Shan

2013-01-01

TAL (transcriptional activator-like) effectors (TALEs) are DNA-binding proteins, containing a modular central domain that recognizes specific DNA sequences. Recently, the crystallographic studies of TALEs revealed the structure of DNA-recognition domain. In this article, molecular dynamics (MD) simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues) with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA), the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL). The conformational analysis of DNA indicates that the 5′ end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism. PMID:24130757

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

PubMed Central

Fowler, Benjamin J.; Gelfand, Bradley D.; Kim, Younghee; Kerur, Nagaraj; Tarallo, Valeria; Hirano, Yoshio; Amarnath, Shoba; Fowler, Daniel H.; Radwan, Marta; Young, Mark T.; Pittman, Keir; Kubes, Paul; Agarwal, Hitesh K.; Parang, Keykavous A.; Hinton, David R.; Bastos-Carvalho, Ana; Li, Shengjian; Yasuma, Tetsuhiro; Mizutani, Takeshi; Yasuma, Reo; Wright, Charles; Ambati, Jayakrishna

2014-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium (RPE) in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease (GVHD), and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases. PMID:25414314

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis.

PubMed

Hara, Yasushi; Hayashi, Kyohei; Nakajima, Takuya; Kagawa, Shizuko; Tazumi, Akihiro; Moore, John E; Matsuda, Motoo

2013-09-01

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

Spinocerebellum Ataxia Type 6: Molecular Mechanisms and Calcium Channel Genetics.

PubMed

Du, Xiaofei; Gomez, Christopher Manuel

2018-01-01

Spinocerebellar ataxia (SCA) type 6 is an autosomal dominant disease affecting cerebellar degeneration. Clinically, it is characterized by pure cerebellar dysfunction, slowly progressive unsteadiness of gait and stance, slurred speech, and abnormal eye movements with late onset. Pathological findings of SCA6 include a diffuse loss of Purkinje cells, predominantly in the cerebellar vermis. Genetically, SCA6 is caused by expansion of a trinucleotide CAG repeat in the last exon of longest isoform CACNA1A gene on chromosome 19p13.1-p13.2. Normal alleles have 4-18 repeats, while alleles causing disease contain 19-33 repeats. Due to presence of a novel internal ribosomal entry site (IRES) with the mRNA, CACNA1A encodes two structurally unrelated proteins with distinct functions within an overlapping open reading frame (ORF) of the same mRNA: (1) α1A subunit of P/Q-type voltage gated calcium channel; (2) α1ACT, a newly recognized transcription factor, with polyglutamine repeat at C-terminal end. Understanding the function of α1ACT in physiological and pathological conditions may elucidate the pathogenesis of SCA6. More importantly, the IRES, as the translational control element of α1ACT, provides a potential therapeutic target for the treatment of SCA6.

Structure prediction of polyglutamine disease proteins: comparison of methods

PubMed Central

2014-01-01

Background The expansion of polyglutamine (poly-Q) repeats in several unrelated proteins is associated with at least ten neurodegenerative diseases. The length of the poly-Q regions plays an important role in the progression of the diseases. The number of glutamines (Q) is inversely related to the onset age of these polyglutamine diseases, and the expansion of poly-Q repeats has been associated with protein misfolding. However, very little is known about the structural changes induced by the expansion of the repeats. Computational methods can provide an alternative to determine the structure of these poly-Q proteins, but it is important to evaluate their performance before large scale prediction work is done. Results In this paper, two popular protein structure prediction programs, I-TASSER and Rosetta, have been used to predict the structure of the N-terminal fragment of a protein associated with Huntington's disease with 17 glutamines. Results show that both programs have the ability to find the native structures, but I-TASSER performs better for the overall task. Conclusions Both I-TASSER and Rosetta can be used for structure prediction of proteins with poly-Q repeats. Knowledge of poly-Q structure may significantly contribute to development of therapeutic strategies for poly-Q diseases. PMID:25080018