Reenacting the birth of an intron

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

Hellsten, Uffe; Aspden, Julie L.; Rio, Donald C.

2011-07-01

An intron is an extended genomic feature whose function requires multiple constrained positions - donor and acceptor splice sites, a branch point, a polypyrimidine tract and suitable splicing enhancers - that may be distributed over hundreds or thousands of nucleotides. New introns are therefore unlikely to emerge by incremental accumulation of functional sub-elements. Here we demonstrate that a functional intron can be created de novo in a single step by a segmental genomic duplication. This experiment recapitulates in vivo the birth of an intron that arose in the ancestral jawed vertebrate lineage nearly half a billion years ago.

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

A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

PubMed

Sano, R; Kuboya, E; Nakajima, T; Takahashi, Y; Takahashi, K; Kubo, R; Kominato, Y; Takeshita, H; Yamao, H; Kishida, T; Isa, K; Ogasawara, K; Uchikawa, M

2015-04-01

We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently. © 2014 International Society of Blood Transfusion.

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

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

Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.

2003-06-01

OAK-B135 In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3 kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae, several other motifs, but not the LFY and WUS binding sites previously identified, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally importantmore » for activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection, but also highlight that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.« less

Homing endonucleases from mobile group I introns: discovery to genome engineering

PubMed Central

2014-01-01

Homing endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity. PMID:24589358

RNA structure in splicing: An evolutionary perspective.

PubMed

Lin, Chien-Ling; Taggart, Allison J; Fairbrother, William G

2016-09-01

Pre-mRNA splicing is a key post-transcriptional regulation process in which introns are excised and exons are ligated together. A novel class of structured intron was recently discovered in fish. Simple expansions of complementary AC and GT dimers at opposite boundaries of an intron were found to form a bridging structure, thereby enforcing correct splice site pairing across the intron. In some fish introns, the RNA structures are strong enough to bypass the need of regulatory protein factors for splicing. Here, we discuss the prevalence and potential functions of highly structured introns. In humans, structured introns usually arise through the co-occurrence of C and G-rich repeats at intron boundaries. We explore the potentially instructive example of the HLA receptor genes. In HLA pre-mRNA, structured introns flank the exons that encode the highly polymorphic β sheet cleft, making the processing of the transcript robust to variants that disrupt splicing factor binding. While selective forces that have shaped HLA receptor are fairly atypical, numerous other highly polymorphic genes that encode receptors contain structured introns. Finally, we discuss how the elevated mutation rate associated with the simple repeats that often compose structured intron can make structured introns themselves rapidly evolving elements.

The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

PubMed

Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

2013-01-01

Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

Patterns and rates of intron divergence between humans and chimpanzees

PubMed Central

Gazave, Elodie; Marqués-Bonet, Tomàs; Fernando, Olga; Charlesworth, Brian; Navarro, Arcadi

2007-01-01

Background Introns, which constitute the largest fraction of eukaryotic genes and which had been considered to be neutral sequences, are increasingly acknowledged as having important functions. Several studies have investigated levels of evolutionary constraint along introns and across classes of introns of different length and location within genes. However, thus far these studies have yielded contradictory results. Results We present the first analysis of human-chimpanzee intron divergence, in which differences in the number of substitutions per intronic site (Ki) can be interpreted as the footprint of different intensities and directions of the pressures of natural selection. Our main findings are as follows: there was a strong positive correlation between intron length and divergence; there was a strong negative correlation between intron length and GC content; and divergence rates vary along introns and depending on their ordinal position within genes (for instance, first introns are more GC rich, longer and more divergent, and divergence is lower at the 3' and 5' ends of all types of introns). Conclusion We show that the higher divergence of first introns is related to their larger size. Also, the lower divergence of short introns suggests that they may harbor a relatively greater proportion of regulatory elements than long introns. Moreover, our results are consistent with the presence of functionally relevant sequences near the 5' and 3' ends of introns. Finally, our findings suggest that other parts of introns may also be under selective constraints. PMID:17309804

Intron open reading frames as mobile elements and evolution of a group I intron.

PubMed

Sellem, C H; Belcour, L

1997-05-01

Group I introns are proposed to have become mobile following the acquisition of open reading frames (ORFs) that encode highly specific DNA endonucleases. This proposal implies that intron ORFs could behave as autonomously mobile entities. This was supported by abundant circumstantial evidence but no experiment of ORF transfer from an ORF-containing intron to its ORF-less counterpart has been described. In this paper we present such experiments, which demonstrate the efficient mobility of the mitochondrial nad1-i4-orf1 between two Podospora strains. The homing of this mobile ORF was accompanied by a bidirectional co-conversion that did not systematically involve the whole intron sequence. Orf1 acquisition would be the most recent step in the evolution of the nad1-i4 intron, which has resulted in many strains of Podospora having an intron with two ORFs (biorfic) and four splicing pathways. We show that two of the splicing events that operate in this biorfic intron, as evidenced by PCR experiments, are generated by a 5'-alternative splice site, which is most probably a remnant of the monoorfic ancestral form of the intron. We propose a sequential evolution model that is consistent with the four organizations of the corresponding nad1 locus that we found among various species of the Pyrenomycete family; these organizations consist of no intron, an intron alone, a monoorfic intron, and a biorfic intron.

Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci.

PubMed

Mills, D A; McKay, L L; Dunny, G M

1996-06-01

Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacteria] group II intron. Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01. Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases. Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria. Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype. These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria.

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

NASA Technical Reports Server (NTRS)

Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

1997-01-01

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Modular space station Phase B extension preliminary performance specification. Volume 2: Project

NASA Technical Reports Server (NTRS)

1971-01-01

The four systems of the modular space station project are described, and the interfaces between this project and the shuttle project, the tracking and data relay satellite project, and an arbitrarily defined experiment project are defined. The experiment project was synthesized from internal experiments, detached research and application modules, and attached research and application modules to derive a set of interface requirements which will support multiple combinations of these elements expected during the modular space station mission. The modular space station project element defines a 6-man orbital program capable of growth to a 12-man orbital program capability. The modular space station project element specification defines the modular space station system, the premission operations support system, the mission operations support system, and the cargo module system and their interfaces.

The role of transposable elements in the evolution of non-mammalian vertebrates and invertebrates

PubMed Central

2010-01-01

Background Transposable elements (TEs) have played an important role in the diversification and enrichment of mammalian transcriptomes through various mechanisms such as exonization and intronization (the birth of new exons/introns from previously intronic/exonic sequences, respectively), and insertion into first and last exons. However, no extensive analysis has compared the effects of TEs on the transcriptomes of mammals, non-mammalian vertebrates and invertebrates. Results We analyzed the influence of TEs on the transcriptomes of five species, three invertebrates and two non-mammalian vertebrates. Compared to previously analyzed mammals, there were lower levels of TE introduction into introns, significantly lower numbers of exonizations originating from TEs and a lower percentage of TE insertion within the first and last exons. Although the transcriptomes of vertebrates exhibit significant levels of exonization of TEs, only anecdotal cases were found in invertebrates. In vertebrates, as in mammals, the exonized TEs are mostly alternatively spliced, indicating that selective pressure maintains the original mRNA product generated from such genes. Conclusions Exonization of TEs is widespread in mammals, less so in non-mammalian vertebrates, and very low in invertebrates. We assume that the exonization process depends on the length of introns. Vertebrates, unlike invertebrates, are characterized by long introns and short internal exons. Our results suggest that there is a direct link between the length of introns and exonization of TEs and that this process became more prevalent following the appearance of mammals. PMID:20525173

The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities

PubMed Central

Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

2013-01-01

Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner. PMID:23505475

De novo insertion of an intron into the mammalian sex determining gene, SRY

PubMed Central

O’Neill, Rachel J. Waugh; Brennan, Francine E.; Delbridge, Margaret L.; Crozier, Ross H.; Graves, Jennifer A. Marshall

1998-01-01

Two theories have been proposed to explain the evolution of introns within eukaryotic genes. The introns early theory, or “exon theory of genes,” proposes that introns are ancient and that recombination within introns provided new exon structure, and thus new genes. The introns late theory, or “insertional theory of introns,” proposes that ancient genes existed as uninterrupted exons and that introns have been introduced during the course of evolution. There is still controversy as to how intron–exon structure evolved and whether the majority of introns are ancient or novel. Although there is extensive evidence in support of the introns early theory, phylogenetic comparisons of several genes indicate recent gain and loss of introns within these genes. However, no example has been shown of a protein coding gene, intronless in its ancestral form, which has acquired an intron in a derived form. The mammalian sex determining gene, SRY, is intronless in all mammals studied to date, as is the gene from which it recently evolved. However, we report here comparisons of genomic and cDNA sequences that now provide evidence of a de novo insertion of an intron into the SRY gene of dasyurid marsupials. This recently (approximately 45 million years ago) inserted sequence is not homologous with known transposable elements. Our data demonstrate that introns may be inserted as spliced units within a developmentally crucial gene without disrupting its function. PMID:9465071

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

Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

PubMed Central

Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

1994-01-01

Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years. PMID:7937917

Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns

PubMed Central

Mohr, Georg; Ghanem, Eman; Lambowitz, Alan M.

2010-01-01

Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelments hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting ∼1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases. PMID:20543989

Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia.

PubMed

Gniadkowski, M; Hemmings-Mieszczak, M; Klahre, U; Liu, H X; Filipowicz, W

1996-02-15

Introns of nuclear pre-mRNAs in dicotyledonous plants, unlike introns in vertebrates or yeast, are distinctly rich in A+U nucleotides and this feature is essential for their processing. In order to define more precisely sequence elements important for intron recognition in plants, we investigated the effects of short insertions, either U-rich or A-rich, on splicing of synthetic introns in transfected protoplast of Nicotiana plumbaginifolia. It was found that insertions of U-rich (sequence UUUUUAU) but not A-rich (AUAAAAA) segments can activate splicing of a GC-rich synthetic infron, and that U-rich segments, or multimers thereof, can function irrespective of the site of insertion within the intron. Insertions of multiple U-rich segments, either at the same or different locations, generally had an additive, stimulatory effect on splicing. Mutational analysis showed that replacement of one or two U residues in the UUUUUAU sequence with A or C residues had only a small effect on splicing, but replacement with G residues was strongly inhibitory. Proteins that interact with fragments of natural and synthetic pre-mRNAs in vitro were identified in nuclear extracts of N.plumbaginifolia by UV cross- linking. The profile of cross-linked plant proteins was considerably less complex than that obtained with a HeLa cell nuclear extract. Two major cross-linkable plant proteins had apparent molecular mass of 50 and 54 kDa and showed affinity for oligouridilates present in synGC introns or for poly(U).

Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia.

PubMed Central

Gniadkowski, M; Hemmings-Mieszczak, M; Klahre, U; Liu, H X; Filipowicz, W

1996-01-01

Introns of nuclear pre-mRNAs in dicotyledonous plants, unlike introns in vertebrates or yeast, are distinctly rich in A+U nucleotides and this feature is essential for their processing. In order to define more precisely sequence elements important for intron recognition in plants, we investigated the effects of short insertions, either U-rich or A-rich, on splicing of synthetic introns in transfected protoplast of Nicotiana plumbaginifolia. It was found that insertions of U-rich (sequence UUUUUAU) but not A-rich (AUAAAAA) segments can activate splicing of a GC-rich synthetic infron, and that U-rich segments, or multimers thereof, can function irrespective of the site of insertion within the intron. Insertions of multiple U-rich segments, either at the same or different locations, generally had an additive, stimulatory effect on splicing. Mutational analysis showed that replacement of one or two U residues in the UUUUUAU sequence with A or C residues had only a small effect on splicing, but replacement with G residues was strongly inhibitory. Proteins that interact with fragments of natural and synthetic pre-mRNAs in vitro were identified in nuclear extracts of N.plumbaginifolia by UV cross- linking. The profile of cross-linked plant proteins was considerably less complex than that obtained with a HeLa cell nuclear extract. Two major cross-linkable plant proteins had apparent molecular mass of 50 and 54 kDa and showed affinity for oligouridilates present in synGC introns or for poly(U). PMID:8604302

Modularization of genetic elements promotes synthetic metabolic engineering.

PubMed

Qi, Hao; Li, Bing-Zhi; Zhang, Wen-Qian; Liu, Duo; Yuan, Ying-Jin

2015-11-15

In the context of emerging synthetic biology, metabolic engineering is moving to the next stage powered by new technologies. Systematical modularization of genetic elements makes it more convenient to engineer biological systems for chemical production or other desired purposes. In the past few years, progresses were made in engineering metabolic pathway using synthetic biology tools. Here, we spotlighted the topic of implementation of modularized genetic elements in metabolic engineering. First, we overviewed the principle developed for modularizing genetic elements and then discussed how the genetic modules advanced metabolic engineering studies. Next, we picked up some milestones of engineered metabolic pathway achieved in the past few years. Last, we discussed the rapid raised synthetic biology field of "building a genome" and the potential in metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative Analysis of Vertebrate Dystrophin Loci Indicate Intron Gigantism as a Common Feature

PubMed Central

Pozzoli, Uberto; Elgar, Greg; Cagliani, Rachele; Riva, Laura; Comi, Giacomo P.; Bresolin, Nereo; Bardoni, Alessandra; Sironi, Manuela

2003-01-01

The human DMD gene is the largest known to date, spanning > 2000 kb on the X chromosome. The gene size is mainly accounted for by huge intronic regions. We sequenced 190 kb of Fugu rubripes (pufferfish) genomic DNA corresponding to the complete dystrophin gene (FrDMD) and provide the first report of gene structure and sequence comparison among dystrophin genomic sequences from different vertebrate organisms. Almost all intron positions and phases are conserved between FrDMD and its mammalian counterparts, and the predicted protein product of the Fugu gene displays 55% identity and 71% similarity to human dystrophin. In analogy to the human gene, FrDMD presents several-fold longer than average intronic regions. Analysis of intron sequences of the human and murine genes revealed that they are extremely conserved in size and that a similar fraction of total intron length is represented by repetitive elements; moreover, our data indicate that intron expansion through repeat accumulation in the two orthologs is the result of independent insertional events. The hypothesis that intron length might be functionally relevant to the DMD gene regulation is proposed and substantiated by the finding that dystrophin intron gigantism is common to the three vertebrate genes. [Supplemental material is available online at www.genome.org.] PMID:12727896

A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

PubMed Central

Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

2017-01-01

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

Invariant U2 snRNA nucleotides form a stem loop to recognize the intron early in splicing

PubMed Central

Perriman, Rhonda; Ares, Manuel

2010-01-01

U2 snRNA-intron branchpoint pairing is a critical step in pre-mRNA recognition by the splicing apparatus, but the mechanism by which these two RNAs engage each other is unknown. Here we identify a new U2 snRNA structure, the branchpoint interaction stem-loop (BSL), that presents the U2 nucleotides that will contact the intron. We provide evidence that the BSL forms prior to interaction with the intron, and is disrupted by the DExD/H protein Prp5p during engagement of the snRNA with the intron. In vitro splicing complex assembly in a BSL-destabilized mutant extract suggests that the BSL is required at a previously unrecognized step between commitment complex and prespliceosome formation. The extreme evolutionary conservation of the BSL suggests it represents an ancient structural solution to the problem of intron branchpoint recognition by dynamic RNA elements that must serve multiple functions at other times during splicing. PMID:20471947

Experimental validation of finite element modelling of a modular metal-on-polyethylene total hip replacement.

PubMed

Hua, Xijin; Wang, Ling; Al-Hajjar, Mazen; Jin, Zhongmin; Wilcox, Ruth K; Fisher, John

2014-07-01

Finite element models are becoming increasingly useful tools to conduct parametric analysis, design optimisation and pre-clinical testing for hip joint replacements. However, the verification of the finite element model is critically important. The purposes of this study were to develop a three-dimensional anatomic finite element model for a modular metal-on-polyethylene total hip replacement for predicting its contact mechanics and to conduct experimental validation for a simple finite element model which was simplified from the anatomic finite element model. An anatomic modular metal-on-polyethylene total hip replacement model (anatomic model) was first developed and then simplified with reasonable accuracy to a simple modular total hip replacement model (simplified model) for validation. The contact areas on the articulating surface of three polyethylene liners of modular metal-on-polyethylene total hip replacement bearings with different clearances were measured experimentally in the Leeds ProSim hip joint simulator under a series of loading conditions and different cup inclination angles. The contact areas predicted from the simplified model were then compared with that measured experimentally under the same conditions. The results showed that the simplification made for the anatomic model did not change the predictions of contact mechanics of the modular metal-on-polyethylene total hip replacement substantially (less than 12% for contact stresses and contact areas). Good agreements of contact areas between the finite element predictions from the simplified model and experimental measurements were obtained, with maximum difference of 14% across all conditions considered. This indicated that the simplification and assumptions made in the anatomic model were reasonable and the finite element predictions from the simplified model were valid. © IMechE 2014.

Targeted Deletion of the Antisilencer/Enhancer (ASE) Element from Intron 1 of the Myelin Proteolipid Protein Gene (Plp1) in Mouse Reveals that the Element Is Dispensable for Plp1 Expression in Brain during Development and Remyelination

PubMed Central

Pereira, Glauber B.; Meng, Fanxue; Kockara, Neriman T.; Yang, Baoli; Wight, Patricia A.

2012-01-01

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. While removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is nonfunctional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. PMID:23157328

Evolutionary and biogeographical implications of degraded LAGLIDADG endonuclease functionality and group I intron occurrence in stony corals (Scleractinia) and mushroom corals (Corallimorpharia).

PubMed

Celis, Juan Sebastián; Edgell, David R; Stelbrink, Björn; Wibberg, Daniel; Hauffe, Torsten; Blom, Jochen; Kalinowski, Jörn; Wilke, Thomas

2017-01-01

Group I introns and homing endonuclease genes (HEGs) are mobile genetic elements, capable of invading target sequences in intron-less genomes. LAGLIDADG HEGs are the largest family of endonucleases, playing a key role in the mobility of group I introns in a process known as 'homing'. Group I introns and HEGs are rare in metazoans, and can be mainly found inserted in the COXI gene of some sponges and cnidarians, including stony corals (Scleractinia) and mushroom corals (Corallimorpharia). Vertical and horizontal intron transfer mechanisms have been proposed as explanations for intron occurrence in cnidarians. However, the central role of LAGLIDADG motifs in intron mobility mechanisms remains poorly understood. To resolve questions regarding the evolutionary origin and distribution of group I introns and HEGs in Scleractinia and Corallimorpharia, we examined intron/HEGs sequences within a comprehensive phylogenetic framework. Analyses of LAGLIDADG motif conservation showed a high degree of degradation in complex Scleractinia and Corallimorpharia. Moreover, the two motifs lack the respective acidic residues necessary for metal-ion binding and catalysis, potentially impairing horizontal intron mobility. In contrast, both motifs are highly conserved within robust Scleractinia, indicating a fully functional endonuclease capable of promoting horizontal intron transference. A higher rate of non-synonymous substitutions (Ka) detected in the HEGs of complex Scleractinia and Corallimorpharia suggests degradation of the HEG, whereas lower Ka rates in robust Scleractinia are consistent with a scenario of purifying selection. Molecular-clock analyses and ancestral inference of intron type indicated an earlier intron insertion in complex Scleractinia and Corallimorpharia in comparison to robust Scleractinia. These findings suggest that the lack of horizontal intron transfers in the former two groups is related to an age-dependent degradation of the endonuclease activity. Moreover, they also explain the peculiar geographical patterns of introns in stony and mushroom corals.

Extensive intron gain in the ancestor of placental mammals

PubMed Central

2011-01-01

Background Genome-wide studies of intron dynamics in mammalian orthologous genes have found convincing evidence for loss of introns but very little for intron turnover. Similarly, large-scale analysis of intron dynamics in a few vertebrate genomes has identified only intron losses and no gains, indicating that intron gain is an extremely rare event in vertebrate evolution. These studies suggest that the intron-rich genomes of vertebrates do not allow intron gain. The aim of this study was to search for evidence of de novo intron gain in domesticated genes from an analysis of their exon/intron structures. Results A phylogenomic approach has been used to analyse all domesticated genes in mammals and chordates that originated from the coding parts of transposable elements. Gain of introns in domesticated genes has been reconstructed on well established mammalian, vertebrate and chordate phylogenies, and examined as to where and when the gain events occurred. The locations, sizes and amounts of de novo introns gained in the domesticated genes during the evolution of mammals and chordates has been analyzed. A significant amount of intron gain was found only in domesticated genes of placental mammals, where more than 70 cases were identified. De novo gained introns show clear positional bias, since they are distributed mainly in 5' UTR and coding regions, while 3' UTR introns are very rare. In the coding regions of some domesticated genes up to 8 de novo gained introns have been found. Intron densities in Eutheria-specific domesticated genes and in older domesticated genes that originated early in vertebrates are lower than those for normal mammalian and vertebrate genes. Surprisingly, the majority of intron gains have occurred in the ancestor of placentals. Conclusions This study provides the first evidence for numerous intron gains in the ancestor of placental mammals and demonstrates that adequate taxon sampling is crucial for reconstructing intron evolution. The findings of this comprehensive study slightly challenge the current view on the evolutionary stasis in intron dynamics during the last 100 - 200 My. Domesticated genes could constitute an excellent system on which to analyse the mechanisms of intron gain in placental mammals. Reviewers: this article was reviewed by Dan Graur, Eugene V. Koonin and Jürgen Brosius. PMID:22112745

Intermediate introns in nuclear genes of euglenids - are they a distinct type?

PubMed

Milanowski, Rafał; Gumińska, Natalia; Karnkowska, Anna; Ishikawa, Takao; Zakryś, Bożena

2016-02-29

Nuclear genes of euglenids contain two major types of introns: conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns. Sequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: two in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene. Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/ -8, -7 and -6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns.

Human intron-encoded Alu RNAs are processed and packaged into Wdr79-associated nucleoplasmic box H/ACA RNPs

PubMed Central

Jády, Beáta E.; Ketele, Amandine; Kiss, Tamás

2012-01-01

Alu repetitive sequences are the most abundant short interspersed DNA elements in the human genome. Full-length Alu elements are composed of two tandem sequence monomers, the left and right Alu arms, both derived from the 7SL signal recognition particle RNA. Since Alu elements are common in protein-coding genes, they are frequently transcribed into pre-mRNAs. Here, we demonstrate that the right arms of nascent Alu transcripts synthesized within pre-mRNA introns are processed into metabolically stable small RNAs. The intron-encoded Alu RNAs, termed AluACA RNAs, are structurally highly reminiscent of box H/ACA small Cajal body (CB) RNAs (scaRNAs). They are composed of two hairpin units followed by the essential H (AnAnnA) and ACA box motifs. The mature AluACA RNAs associate with the four H/ACA core proteins: dyskerin, Nop10, Nhp2, and Gar1. Moreover, the 3′ hairpin of AluACA RNAs carries two closely spaced CB localization motifs, CAB boxes (UGAG), which bind Wdr79 in a cumulative fashion. In contrast to canonical H/ACA scaRNPs, which concentrate in CBs, the AluACA RNPs accumulate in the nucleoplasm. Identification of 348 human AluACA RNAs demonstrates that intron-encoded AluACA RNAs represent a novel, large subgroup of H/ACA RNAs, which are apparently confined to human or primate cells. PMID:22892240

Modular Approach to Structural Simulation for Vehicle Crashworthiness Prediction

DOT National Transportation Integrated Search

1975-03-01

A modular formulation for simulation of the structural deformation and deceleration of a vehicle for crashworthiness and collision compatibility is presented. This formulation includes three dimensional beam elements, various spring elements, rigid b...

Localization of a bacterial group II intron-encoded protein in human cells.

PubMed

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

2015-08-05

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

Localization of a bacterial group II intron-encoded protein in human cells

PubMed Central

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

2015-01-01

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

The splicing of tiny introns of Paramecium is controlled by MAGO.

PubMed

Contreras, Julia; Begley, Victoria; Marsella, Laura; Villalobo, Eduardo

2018-07-15

The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size. Copyright © 2018 Elsevier B.V. All rights reserved.

Mechanism for DNA transposons to generate introns on genomic scales

PubMed Central

Huff, Jason T.; Zilberman, Daniel; Roy, Scott W.

2017-01-01

Discovered four decades ago, the existence of introns was one of the most unexpected findings in molecular biology1. Introns are sequences interrupting genes that must be removed as part of mRNA production. Genome sequencing projects have documented that most eukaryotic genes contain at least one and frequently many introns2,3. Comparison of these genomes reveals a history of long evolutionary periods with little intron gain punctuated by episodes of rapid, extensive gain2,3. However, no detailed mechanism for such episodic intron generation has been empirically supported on a sufficient scale, despite several proposals4–8. Here we show how short non-autonomous DNA transposons independently generated hundreds to thousands of introns in the prasinophyte Micromonas pusilla and the pelagophyte Aureococcus anophagefferens. Each transposon carries one splice site. The other splice site is co-opted from gene sequence duplicated upon transposon insertion, allowing perfect splicing out of RNA. The distributions of sequences that can be co-opted are biased with respect to codons, and phasing of transposon-generated introns is similarly biased. These transposons insert between preexisting nucleosomes, so that multiple nearby insertions generate nucleosome-sized intervening segments. Thus, transposon insertion and sequence co-option may explain the intron phase biases2 and prevalence of nucleosome-sized exons9 observed in eukaryotes. Overall, the two independent examples of proliferating elements illustrate a general DNA transposon mechanism plausibly accounting for episodes of rapid, extensive intron gain during eukaryotic evolution2,3. PMID:27760113

Post-transcriptional regulation mediated by specific neurofilament introns in vivo.

PubMed

Wang, Chen; Szaro, Ben G

2016-04-01

Neurons regulate genes post-transcriptionally to coordinate the supply of cytoskeletal proteins, such as the medium neurofilament (NEFM), with demand for structural materials in response to extracellular cues encountered by developing axons. By using a method for evaluating functionality of cis-regulatory gene elements in vivo through plasmid injection into Xenopus embryos, we discovered that splicing of a specific nefm intron was required for robust transgene expression, regardless of promoter or cell type. Transgenes utilizing the nefm 3'-UTR but substituting other nefm introns expressed little or no protein owing to defects in handling of the messenger (m)RNA as opposed to transcription or splicing. Post-transcriptional events at multiple steps, but mainly during nucleocytoplasmic export, contributed to these varied levels of protein expression. An intron of the β-globin gene was also able to promote expression in a manner identical to that of the nefm intron, implying a more general preference for certain introns in controlling nefm expression. These results expand our knowledge of intron-mediated gene expression to encompass neurofilaments, indicating an additional layer of complexity in the control of a cytoskeletal gene needed for developing and maintaining healthy axons. © 2016. Published by The Company of Biologists Ltd.

Genomic structure of two ras family genes in the slime mold Physarum polycephalum.

PubMed

Trzcińska-Danielewicz, Joanna; Kozlowski, Piotr; Gierdal, Katarzyna; Wiejak, Jolanta; Jagielski, Adam; Toczko, Kazimierz; Fronk, Jan

2002-08-01

Genomic structure of two Physarum polycephalum ras family genes, Ppras2 and Pprap1, has been determined, including the upstream region of the latter. The genes are interrupted by three and four introns, respectively. The first intron of Ppras2 has the same location within the coding sequence as the first intron in another ras homolog from this organism, Ppras1 [Trzcińska-Danielewicz, J., Kozlowski, P., and Toczko, K. (1996). "Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene", Gene 169, pp. 143-144]. All introns, ranging from 53 to ca. 460 base pairs, have the canonical 5' and 3' ends, are greatly enriched in pyrimidines in the coding strand and have frequent pyrimidines-only tracts. These latter features seem to be responsible for the difficulties in cloning and sequencing of parts of these genes. Short sequences shared with P. polycephalum transposon-like repeats are common in the introns, indicating a possible role of transposition in intron evolution. In all three ras family genes phase zero introns are located mostly between sequences coding for regular protein secondary structure elements.

Spliceosomal Intron Insertions in Genome Compacted Ray-Finned Fishes as Evident from Phylogeny of MC Receptors, Also Supported by a Few Other GPCRs

PubMed Central

Sinha, Rahul; Goyal, Pankaj; Grapputo, Alessandro

2011-01-01

Background Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution. Methodology/Principal Findings We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes. Conclusions/Significance The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality. PMID:21850219

Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination.

PubMed

Pereira, Glauber B; Meng, Fanxue; Kockara, Neriman T; Yang, Baoli; Wight, Patricia A

2013-02-01

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is non-functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. © 2012 International Society for Neurochemistry.

The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate?

PubMed Central

Koonin, Eugene V

2006-01-01

Background Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes. Results I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the α-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron-rich genome. Thus, it appears that ancestors of spliceosomal introns, indeed, have existed since the earliest stages of life's evolution, in a formal agreement with the introns-early scenario. However, there is no evidence that these ancient introns ever became widespread before the emergence of eukaryotes, hence, the central tenet of introns-early, the role of introns in early evolution of proteins, has no support. However, the demonstration that numerous introns invaded eukaryotic genes at the outset of eukaryotic evolution and that subsequent intron gain has been limited in many eukaryotic lineages implicates introns as an ancestral feature of eukaryotic genomes and refutes radical versions of introns-late. Perhaps, most importantly, I argue that the intron invasion triggered other pivotal events of eukaryogenesis, including the emergence of the spliceosome, the nucleus, the linear chromosomes, the telomerase, and the ubiquitin signaling system. This concept of eukaryogenesis, in a sense, revives some tenets of the exon hypothesis, by assigning to introns crucial roles in eukaryotic evolutionary innovation. Conclusion The scenario of the origin and evolution of introns that is best compatible with the results of comparative genomics and theoretical considerations goes as follows: self-splicing introns since the earliest stages of life's evolution – numerous spliceosomal introns invading genes of the emerging eukaryote during eukaryogenesis – subsequent lineage-specific loss and gain of introns. The intron invasion, probably, spawned by the mitochondrial endosymbiont, might have critically contributed to the emergence of the principal features of the eukaryotic cell. This scenario combines aspects of the introns-early and introns-late views. Reviewers this article was reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole. PMID:16907971

Cooperative action of multiple cis-acting elements is required for N-myc expression in branchial arches: specific contribution of GATA3.

PubMed

Potvin, Eric; Beuret, Laurent; Cadrin-Girard, Jean-François; Carter, Marcelle; Roy, Sophie; Tremblay, Michel; Charron, Jean

2010-11-01

The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb -8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.

The legumin gene family: structure of a B type gene of Vicia faba and a possible legumin gene specific regulatory element.

PubMed Central

Bäumlein, H; Wobus, U; Pustell, J; Kafatos, F C

1986-01-01

The field bean, Vicia faba L. var. minor, possesses two sub-families of 11 S legumin genes named A and B. We isolated from a genomic library a B-type gene (LeB4) and determined its primary DNA sequence. Gene LeB4 codes for a 484 amino acid residue prepropolypeptide, encompassing a signal peptide of 22 amino acid residues, an acidic, very hydrophilic alpha-chain of 281 residues and a basic, somewhat hydrophobic beta-chain of 181 residues. The latter two coding regions are immediately contiguous, but each is interrupted by a short intron. Type A legumin genes from soybean and pea are known to have introns in the same two positions, in addition to an extra intron (within the alpha-coding sequence). Sequence comparisons of legumin genes from these three plants revealed a highly conserved sequence element of at least 28 bp, centered at approximately 100 bp upstream of each cap site. The element is absent from the equivalent position of all non-legumin and other plant and fungal genes examined. We tentatively name this element "legumin box" and suggest that it may have a function in the regulation of legumin gene expression. PMID:3960730

Conserved Nonexonic Elements: A Novel Class of Marker for Phylogenomics.

PubMed

Edwards, Scott V; Cloutier, Alison; Baker, Allan J

2017-11-01

Noncoding markers have a particular appeal as tools for phylogenomic analysis because, at least in vertebrates, they appear less subject to strong variation in GC content among lineages. Thus far, ultraconserved elements (UCEs) and introns have been the most widely used noncoding markers. Here we analyze and study the evolutionary properties of a new type of noncoding marker, conserved nonexonic elements (CNEEs), which consists of noncoding elements that are estimated to evolve slower than the neutral rate across a set of species. Although they often include UCEs, CNEEs are distinct from UCEs because they are not ultraconserved, and, most importantly, the core region alone is analyzed, rather than both the core and its flanking regions. Using a data set of 16 birds plus an alligator outgroup, and ∼3600-∼3800 loci per marker type, we found that although CNEEs were less variable than bioinformatically derived UCEs or introns and in some cases exhibited a slower approach to branch resolution as determined by phylogenomic subsampling, the quality of CNEE alignments was superior to those of the other markers, with fewer gaps and missing species. Phylogenetic resolution using coalescent approaches was comparable among the three marker types, with most nodes being fully and congruently resolved. Comparison of phylogenetic results across the three marker types indicated that one branch, the sister group to the passerine + falcon clade, was resolved differently and with moderate (>70%) bootstrap support between CNEEs and UCEs or introns. Overall, CNEEs appear to be promising as phylogenomic markers, yielding phylogenetic resolution as high as for UCEs and introns but with fewer gaps, less ambiguity in alignments and with patterns of nucleotide substitution more consistent with the assumptions of commonly used methods of phylogenetic analysis. © The Author(s) 2017. Published by Oxford University Press on behalf of the Systematic Biologists.

Conserved Nonexonic Elements: A Novel Class of Marker for Phylogenomics

PubMed Central

Cloutier, Alison; Baker, Allan J.

2017-01-01

Abstract Noncoding markers have a particular appeal as tools for phylogenomic analysis because, at least in vertebrates, they appear less subject to strong variation in GC content among lineages. Thus far, ultraconserved elements (UCEs) and introns have been the most widely used noncoding markers. Here we analyze and study the evolutionary properties of a new type of noncoding marker, conserved nonexonic elements (CNEEs), which consists of noncoding elements that are estimated to evolve slower than the neutral rate across a set of species. Although they often include UCEs, CNEEs are distinct from UCEs because they are not ultraconserved, and, most importantly, the core region alone is analyzed, rather than both the core and its flanking regions. Using a data set of 16 birds plus an alligator outgroup, and ∼3600–∼3800 loci per marker type, we found that although CNEEs were less variable than bioinformatically derived UCEs or introns and in some cases exhibited a slower approach to branch resolution as determined by phylogenomic subsampling, the quality of CNEE alignments was superior to those of the other markers, with fewer gaps and missing species. Phylogenetic resolution using coalescent approaches was comparable among the three marker types, with most nodes being fully and congruently resolved. Comparison of phylogenetic results across the three marker types indicated that one branch, the sister group to the passerine + falcon clade, was resolved differently and with moderate (>70%) bootstrap support between CNEEs and UCEs or introns. Overall, CNEEs appear to be promising as phylogenomic markers, yielding phylogenetic resolution as high as for UCEs and introns but with fewer gaps, less ambiguity in alignments and with patterns of nucleotide substitution more consistent with the assumptions of commonly used methods of phylogenetic analysis. PMID:28637293

Insights into the history of a bacterial group II intron remnant from the genomes of the nitrogen-fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae.

PubMed

Toro, N; Martínez-Rodríguez, L; Martínez-Abarca, F

2014-10-01

Group II introns are self-splicing catalytic RNAs that act as mobile retroelements. In bacteria, they are thought to be tolerated to some extent because they self-splice and home preferentially to sites outside of functional genes, generally within intergenic regions or in other mobile genetic elements, by mechanisms including the divergence of DNA target specificity to prevent target site saturation. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti and was first described in the GR4 strain. Like other bacterial group II introns, RmInt1 tends to evolve toward an inactive form by fragmentation, with loss of the 3' terminus. We identified genomic evidence of a fragmented intron closely related to RmInt1 buried in the genome of the extant S. meliloti/S. medicae species. By studying this intron, we obtained evidence for the occurrence of intron insertion before the divergence of ancient rhizobial species. This fragmented group II intron has thus existed for a long time and has provided sequence variation, on which selection can act, contributing to diverse genetic rearrangements, and to generate pan-genome divergence after strain differentiation. The data presented here suggest that fragmented group II introns within intergenic regions closed to functionally important neighboring genes may have been microevolutionary forces driving adaptive evolution of these rhizobial species.

Insights into the history of a bacterial group II intron remnant from the genomes of the nitrogen-fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

PubMed Central

Toro, N; Martínez-Rodríguez, L; Martínez-Abarca, F

2014-01-01

Group II introns are self-splicing catalytic RNAs that act as mobile retroelements. In bacteria, they are thought to be tolerated to some extent because they self-splice and home preferentially to sites outside of functional genes, generally within intergenic regions or in other mobile genetic elements, by mechanisms including the divergence of DNA target specificity to prevent target site saturation. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti and was first described in the GR4 strain. Like other bacterial group II introns, RmInt1 tends to evolve toward an inactive form by fragmentation, with loss of the 3′ terminus. We identified genomic evidence of a fragmented intron closely related to RmInt1 buried in the genome of the extant S. meliloti/S. medicae species. By studying this intron, we obtained evidence for the occurrence of intron insertion before the divergence of ancient rhizobial species. This fragmented group II intron has thus existed for a long time and has provided sequence variation, on which selection can act, contributing to diverse genetic rearrangements, and to generate pan-genome divergence after strain differentiation. The data presented here suggest that fragmented group II introns within intergenic regions closed to functionally important neighboring genes may have been microevolutionary forces driving adaptive evolution of these rhizobial species. PMID:24736785

Multiple recent horizontal transfers of the cox1 intron in Solanaceae and extended co-conversion of flanking exons

PubMed Central

2011-01-01

Background The most frequent case of horizontal transfer in plants involves a group I intron in the mitochondrial gene cox1, which has been acquired via some 80 separate plant-to-plant transfer events among 833 diverse angiosperms examined. This homing intron encodes an endonuclease thought to promote the intron's promiscuous behavior. A promising experimental approach to study endonuclease activity and intron transmission involves somatic cell hybridization, which in plants leads to mitochondrial fusion and genome recombination. However, the cox1 intron has not yet been found in the ideal group for plant somatic genetics - the Solanaceae. We therefore undertook an extensive survey of this family to find members with the intron and to learn more about the evolutionary history of this exceptionally mobile genetic element. Results Although 409 of the 426 species of Solanaceae examined lack the cox1 intron, it is uniformly present in three phylogenetically disjunct clades. Despite strong overall incongruence of cox1 intron phylogeny with angiosperm phylogeny, two of these clades possess nearly identical intron sequences and are monophyletic in intron phylogeny. These two clades, and possibly the third also, contain a co-conversion tract (CCT) downstream of the intron that is extended relative to all previously recognized CCTs in angiosperm cox1. Re-examination of all published cox1 genes uncovered additional cases of extended co-conversion and identified a rare case of putative intron loss, accompanied by full retention of the CCT. Conclusions We infer that the cox1 intron was separately and recently acquired by at least three different lineages of Solanaceae. The striking identity of the intron and CCT from two of these lineages suggests that one of these three intron captures may have occurred by a within-family transfer event. This is consistent with previous evidence that horizontal transfer in plants is biased towards phylogenetically local events. The discovery of extended co-conversion suggests that other cox1 conversions may be longer than realized but obscured by the exceptional conservation of plant mitochondrial sequences. Our findings provide further support for the rampant-transfer model of cox1 intron evolution and recommend the Solanaceae as a model system for the experimental analysis of cox1 intron transfer in plants. PMID:21943226

The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells

PubMed Central

Barnea-Yizhar, Ofer; Ram, Sigal; Kovalev, Ekaterina; Azriel, Aviva; Rand, Ulfert; Nakayama, Manabu; Hauser, Hansjörg; Gepstein, Lior; Levi, Ben-Zion

2016-01-01

Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron’s properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation. PMID:27257682

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron.

PubMed

Emblem, Åse; Karlsen, Bård Ove; Evertsen, Jussi; Johansen, Steinar D

2011-11-01

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3' end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals. Copyright © 2011 Elsevier Inc. All rights reserved.

Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites in C. elegans

PubMed Central

Ragle, James Matthew; Katzman, Sol; Akers, Taylor F.; Barberan-Soler, Sergio; Zahler, Alan M.

2015-01-01

Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3′ splice site (furthest from the 5′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3′ splice site (closer to the 5′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. PMID:25922281

Modular assembly of optical nanocircuits.

PubMed

Shi, Jinwei; Monticone, Francesco; Elias, Sarah; Wu, Yanwen; Ratchford, Daniel; Li, Xiaoqin; Alù, Andrea

2014-05-29

A key element enabling the microelectronic technology advances of the past decades has been the conceptualization of complex circuits with versatile functionalities as being composed of the proper combination of basic 'lumped' circuit elements (for example, inductors and capacitors). In contrast, modern nanophotonic systems are still far from a similar level of sophistication, partially because of the lack of modularization of their response in terms of basic building blocks. Here we demonstrate the design, assembly and characterization of relatively complex photonic nanocircuits by accurately positioning a number of metallic and dielectric nanoparticles acting as modular lumped elements. The nanoparticle clusters produce the desired spectral response described by simple circuit rules and are shown to be dynamically reconfigurable by modifying the direction or polarization of impinging signals. Our work represents an important step towards extending the powerful modular design tools of electronic circuits into nanophotonic systems.

Modular assembly of optical nanocircuits

NASA Astrophysics Data System (ADS)

Shi, Jinwei; Monticone, Francesco; Elias, Sarah; Wu, Yanwen; Ratchford, Daniel; Li, Xiaoqin; Alù, Andrea

2014-05-01

A key element enabling the microelectronic technology advances of the past decades has been the conceptualization of complex circuits with versatile functionalities as being composed of the proper combination of basic ‘lumped’ circuit elements (for example, inductors and capacitors). In contrast, modern nanophotonic systems are still far from a similar level of sophistication, partially because of the lack of modularization of their response in terms of basic building blocks. Here we demonstrate the design, assembly and characterization of relatively complex photonic nanocircuits by accurately positioning a number of metallic and dielectric nanoparticles acting as modular lumped elements. The nanoparticle clusters produce the desired spectral response described by simple circuit rules and are shown to be dynamically reconfigurable by modifying the direction or polarization of impinging signals. Our work represents an important step towards extending the powerful modular design tools of electronic circuits into nanophotonic systems.

A functional study of proximal goat β-casein promoter and intron 1 in immortalized goat mammary epithelial cells.

PubMed

Kung, M H; Lee, Y J; Hsu, J T; Huang, M C; Ju, Y T

2015-06-01

Goat β-casein (CSN2) promoter has been extensively used to derive expression of recombinant therapeutic protein in transgenic goats; however, little direct evidence exists for signaling molecules and the cis-elements of goat CSN2 promoter in response to lactogenic hormone stimulation in goat mammary epithelial cells. Here, we use an immortalized caprine mammary epithelial cell line (CMC) to search for evidence of the above. Serial 5'-flanking regions deleted of promoter and intron 1 in goat CSN2 (-4,047 to +2,054) driven by firefly luciferase reporter gene were constructed and applied to measure promoter activity in CMC. The intron 1 region (+393 to +501) significantly decreased basal activity of the promoter. This finding contradicts other studies of the role of intron 1. The signal transducer and activator of transcription (STAT)5a played a significant role in activating promoter activity by prolactin stimulation. Hydrocortisone enhanced and prolonged the activity of STAT5a and promoter in CMC, but was independent of the glucocorticoid receptor response element. The minimum length of the CSN2 promoter segment in response to lactogenic stimulation was confirmed by 5' serial deletions. A cis-element located from -300 to -90 in proximal goat CSN2 promoter that is absent in bovine and human CSN2 promoter was newly identified. We demonstrated the presence of a STAT5a binding site (-102 to -82) and preservation of the guanosine nucleotide at position -90 based on responses to the presence of lactogenic hormone using internal deletions and point mutations of the predicted STAT5a binding site, and chromatin immunoprecipitation assay. Together, these findings demonstrate that the proximal -300 bp of goat CSN2 promoter containing the STAT5a binding site (-102 to -82) is the response element for lactogenic hormone stimulation. Additionally, intron 1 may be required for tissue or developmental stage-specific expression in mammary gland. The role of the far-distal regions of goat CSN2 promoter in high-level lactogenic hormone induction and specific expression require further examination. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

ABO alleles are linked with haplotypes of an erythroid cell-specific regulatory element in intron 1 with a few exceptions attributable to genetic recombination.

PubMed

Nakajima, T; Sano, R; Takahashi, Y; Watanabe, K; Kubo, R; Kobayashi, M; Takahashi, K; Takeshita, H; Kominato, Y

2016-01-01

Recent investigation of transcriptional regulation of the ABO genes has identified a candidate erythroid cell-specific regulatory element, named the +5·8-kb site, in the first intron of ABO. Six haplotypes of the site have been reported previously. The present genetic population study demonstrated that each haplotype was mostly linked with specific ABO alleles with a few exceptions, possibly as a result of hybrid formation between common ABO alleles. Thus, investigation of these haplotypes could provide a clue to further elucidation of ABO alleles. © 2015 International Society of Blood Transfusion.

Portable modular detection system

DOEpatents

Brennan, James S [Rodeo, CA; Singh, Anup [Danville, CA; Throckmorton, Daniel J [Tracy, CA; Stamps, James F [Livermore, CA

2009-10-13

Disclosed herein are portable and modular detection devices and systems for detecting electromagnetic radiation, such as fluorescence, from an analyte which comprises at least one optical element removably attached to at least one alignment rail. Also disclosed are modular detection devices and systems having an integrated lock-in amplifier and spatial filter and assay methods using the portable and modular detection devices.

Identification of an Intronic Splicing Enhancer Essential for the Inclusion of FGFR2 Exon IIIc*S⃞

PubMed Central

Seth, Puneet; Miller, Heather B.; Lasda, Erika L.; Pearson, James L.; Garcia-Blanco, Mariano A.

2008-01-01

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human β-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5′-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements. PMID:18256031

Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock.

PubMed

Freitas, F Zanolli; Bertolini, M C

2004-12-01

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase ( gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30 degrees C to 45 degrees C). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans -acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.

Bipolar localization of the group II intron Ll.LtrB is maintained in Escherichia coli deficient in nucleoid condensation, chromosome partitioning and DNA replication.

PubMed

Beauregard, Arthur; Chalamcharla, Venkata R; Piazza, Carol Lyn; Belfort, Marlene; Coros, Colin J

2006-11-01

Group II introns are mobile genetic elements that invade their cognate intron-minus alleles via an RNA intermediate, in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. In Escherichia coli, retrotransposition of the lactococcal group II intron, Ll.LtrB, occurs preferentially within the Ori and Ter macrodomains of the E. coli chromosome. These macrodomains migrate towards the poles of the cell, where the intron-encoded protein, LtrA, localizes. Here we investigate whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron integration and LtrA distribution in E. coli. We thus examined these properties in the absence of the nucleoid-associated proteins H-NS, StpA and MukB, in variants of partitioning functions including the centromere-like sequence migS and the actin homologue MreB, as well as in the replication mutants DeltaoriC, seqA, tus and topoIV (ts). Although there were some dramatic fluctuations in retrotransposition levels in these hosts, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid-free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron-encoded protein at the poles of the cell.

Effects of pre-existing anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine.

PubMed

Chuan, Yap P; Rivera-Hernandez, Tania; Wibowo, Nani; Connors, Natalie K; Wu, Yang; Hughes, Fiona K; Lua, Linda H L; Middelberg, Anton P J

2013-09-01

Modularization of a peptide antigen for presentation on a microbially synthesized murine polyomavirus (MuPyV) virus-like particle (VLP) offers a new alternative for rapid and low-cost vaccine delivery at a global scale. In this approach, heterologous modules containing peptide antigenic elements are fused to and displayed on the VLP carrier, allowing enhancement of peptide immunogenicity via ordered and densely repeated presentation of the modules. This study addresses two key engineering questions pertaining to this platform, exploring the effects of (i) pre-existing carrier-specific immunity on modular VLP vaccine effectiveness and (ii) increase in the antigenic element number per VLP on peptide-specific immune response. These effects were studied in a mouse model and with modular MuPyV VLPs presenting a group A streptococcus (GAS) peptide antigen, J8i. The data presented here demonstrate that immunization with a modular VLP could induce high levels of J8i-specific antibodies despite a strong pre-existing anti-carrier immune response. Doubling of the J8i antigenic element number per VLP did not enhance J8i immunogenicity at a constant peptide dose. However, the strategy, when used in conjunction with increased VLP dose, could effectively increase the peptide dose up to 10-fold, leading to a significantly higher J8i-specific antibody titer. This study further supports feasibility of the MuPyV modular VLP vaccine platform by showing that, in the absence of adjuvant, modularized GAS antigenic peptide at a dose as low as 150 ng was sufficient to raise a high level of peptide-specific IgGs indicative of bactericidal activity. Copyright © 2013 Wiley Periodicals, Inc.

Permanent Neonatal Diabetes Caused by Creation of an Ectopic Splice Site within the INS Gene

PubMed Central

Gastaldo, Elena; Harries, Lorna W.; Rubio-Cabezas, Oscar; Castaño, Luis

2012-01-01

Background The aim of this study was to characterize the genetic etiology in a patient who presented with permanent neonatal diabetes at 2 months of age. Methodology/Principal Findings Regulatory elements and coding exons 2 and 3 of the INS gene were amplified and sequenced from genomic and complementary DNA samples. A novel heterozygous INS mutation within the terminal intron of the gene was identified in the proband and her affected father. This mutation introduces an ectopic splice site leading to the insertion of 29 nucleotides from the intronic sequence into the mature mRNA, which results in a longer and abnormal transcript. Conclusions/Significance This study highlights the importance of routinely sequencing the exon-intron boundaries and the need to carry out additional studies to confirm the pathogenicity of any identified intronic genetic variants. PMID:22235272

Modular support blocks for fluid lines

NASA Technical Reports Server (NTRS)

Dimino, J. M.; Deskin, R. D.

1974-01-01

Modular line block comprises matched modular elements machined to accept fluid lines of different diameters. Modules can support different fluid-line configurations. Top and bottom surfaces are machined to accept dovetail strip used for holding modules together. End modules have holes drilled through to accept fastening screws.

Pea chloroplast tRNA(Lys) (UUU) gene: transcription and analysis of an intron-containing gene.

PubMed

Boyer, S K; Mullet, J E

1988-07-01

The pea chloroplast trnK gene which encodes tRNA(Lys) (UUU) was sequenced. TrnK is located 210 bp upstream from the promoter of psbA and immediately downstream from the 3'-end of rbcL. The gene is transcribed from the same DNA strand as psbA and rbcL. A 2447 bp intron with class II features is located in the trnK anticodon loop. The intron contains a 506 amino acid open reading frame which could encode an RNA maturase. The primary transcript of trnK is 2.9 kb long; its 5'-end was identified as a site of transcription initiation by in vitro transcription experiments. The 5'-terminus is adjacent to DNA sequences previously identified as transcription promoter elements. The most abundant trnK transcript is 2.5 kb long with termini corresponding to the 5' and 3' ends of the trnK exons. Intron specific RNAs were not detected. This suggests that RNA processing which produces tRNA(Lys) leads to rapid degradation of intron sequences.

Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene.

PubMed

Gonçalves, Ana; Oliveira, Jorge; Coelho, Teresa; Taipa, Ricardo; Melo-Pires, Manuel; Sousa, Mário; Santos, Rosário

2017-10-03

A broad mutational spectrum in the dystrophin ( DMD ) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD , adding to the diversity of mutational events that give rise to D/BMD.

Modular optical detector system

DOEpatents

Horn, Brent A [Livermore, CA; Renzi, Ronald F [Tracy, CA

2006-02-14

A modular optical detector system. The detector system is designed to detect the presence of molecules or molecular species by inducing fluorescence with exciting radiation and detecting the emitted fluorescence. Because the system is capable of accurately detecting and measuring picomolar concentrations it is ideally suited for use with microchemical analysis systems generally and capillary chromatographic systems in particular. By employing a modular design, the detector system provides both the ability to replace various elements of the detector system without requiring extensive realignment or recalibration of the components as well as minimal user interaction with the system. In addition, the modular concept provides for the use and addition of a wide variety of components, including optical elements (lenses and filters), light sources, and detection means, to fit particular needs.

Modular Power Standard for Space Explorations Missions

NASA Technical Reports Server (NTRS)

Oeftering, Richard C.; Gardner, Brent G.

2016-01-01

Future human space exploration will most likely be composed of assemblies of multiple modular spacecraft elements with interconnected electrical power systems. An electrical system composed of a standardized set modular building blocks provides significant development, integration, and operational cost advantages. The modular approach can also provide the flexibility to configure power systems to meet the mission needs. A primary goal of the Advanced Exploration Systems (AES) Modular Power System (AMPS) project is to establish a Modular Power Standard that is needed to realize these benefits. This paper is intended to give the space exploration community a "first look" at the evolving Modular Power Standard and invite their comments and technical contributions.

Modular space station, phase B extension. Program operations plan

NASA Technical Reports Server (NTRS)

1971-01-01

An organized approach is defined for establishing the most significant requirements pertaining to mission operations, information management, and computer program design and development for the modular space station program. The operations plan pertains to the space station and experiment module program elements and to the ground elements required for mission management and mission support operations.

Structural features of diverse Pin-II proteinase inhibitor genes from Capsicum annuum.

PubMed

Mahajan, Neha S; Dewangan, Veena; Lomate, Purushottam R; Joshi, Rakesh S; Mishra, Manasi; Gupta, Vidya S; Giri, Ashok P

2015-02-01

The proteinase inhibitor (PI) genes from Capsicum annuum were characterized with respect to their UTR, introns and promoter elements. The occurrence of PIs with circularly permuted domain organization was evident. Several potato inhibitor II (Pin-II) type proteinase inhibitor (PI) genes have been analyzed from Capsicum annuum (L.) with respect to their differential expression during plant defense response. However, complete gene characterization of any of these C. annuum PIs (CanPIs) has not been carried out so far. Complete gene architectures of a previously identified CanPI-7 (Beads-on-string, Type A) and a member of newly isolated Bracelet type B, CanPI-69 are reported in this study. The 5' UTR (untranslated region), 3'UTR, and intronic sequences of both the CanPI genes were obtained. The genomic sequence of CanPI-7 exhibited, exon 1 (49 base pair, bp) and exon 2 (740 bp) interrupted by a 294-bp long type I intron. We noted the occurrence of three multi-domain PIs (CanPI-69, 70, 71) with circularly permuted domain organization. CanPI-69 was found to possess exon 1 (49 bp), exon 2 (551 bp) and a 584-bp long type I intron. The upstream sequence analysis of CanPI-7 and CanPI-69 predicted various transcription factor-binding sites including TATA and CAAT boxes, hormone-responsive elements (ABRELATERD1, DOFCOREZM, ERELEE4), and a defense-responsive element (WRKY71OS). Binding of transcription factors such as zinc finger motif MADS-box and MYB to the promoter regions was confirmed using electrophoretic mobility shift assay followed by mass spectrometric identification. The 3' UTR analysis for 25 CanPI genes revealed unique/distinct 3' UTR sequence for each gene. Structures of three domain CanPIs of type A and B were predicted and further analyzed for their attributes. This investigation of CanPI gene architecture will enable the better understanding of the genetic elements present in CanPIs.

The human myelin oligodendrocyte glycoprotein (MOG) gene: Complete nucleotide sequence and structural characterization

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

Paule Roth, M.; Malfroy, L.; Offer, C.

1995-07-20

Human myelin oligodendrocyte glycoprotein (MOG), a myelin component of the central nervous system, is a candidate target antigen for autoimmune-mediated demyelination. We have isolated and sequenced part of a cosmid clone that contains the entire human MOG gene. The primary nuclear transcript, extending from the putative start of transcription to the site of poly(A) addition, is 15,561 nucleotides in length. The human MOG gene contains 8 exons, separated by 7 introns; canonical intron/exon boundary sites are observed at each junction. The introns vary in size from 242 to 6484 bp and contain numerous repetitive DNA elements, including 14 Alu sequencesmore » within 3 introns. Another Alu element is located in the 3{prime}-untranslated region of the gene. Alu sequences were classified with respect to subfamily assignment. Seven hundred sixty-three nucleotides 5{prime} of the transcription start and 1214 nucleotides 3{prime} of the poly(A) addition sites were also sequenced. The 5{prime}-flanking region revealed the presence of several consensus sequences that could be relevant in the transcription of the MOG gene, in particular binding sites in common with other myelin gene promoters. Two polymorphic intragenic dinucleotide (CA){sub n} and tetranucleotide (TAAA){sub n} repeats were identified and may provide genetic marker tools for association and linkage studies. 50 refs., 3 figs., 3 tabs.« less

Numerical simulations of human tibia osteosynthesis using modular plates based on Nitinol staples.

PubMed

Tarniţă, Daniela; Tarniţă, D N; Popa, D; Grecu, D; Tarniţă, Roxana; Niculescu, D; Cismaru, F

2010-01-01

The shape memory alloys exhibit a number of remarkable properties, which open new possibilities in engineering and more specifically in biomedical engineering. The most important alloy used in biomedical applications is NiTi. This alloy combines the characteristics of the shape memory effect and superelasticity with excellent corrosion resistance, wear characteristics, mechanical properties and a good biocompatibility. These properties make it an ideal biological engineering material, especially in orthopedic surgery and orthodontics. In this work, modular plates for the osteosynthesis of the long bones fractures are presented. The proposed modular plates are realized from identical modules, completely interchangeable, made of titanium or stainless steel having as connecting elements U-shaped staples made of Nitinol. Using computed tomography (CT) images to provide three-dimensional geometric details and SolidWorks software package, the three dimensional virtual models of the tibia bone and of the modular plates are obtained. The finite element models of the tibia bone and of the modular plate are generated. For numerical simulation, VisualNastran software is used. Finally, displacements diagram, von Misses strain diagram, for the modular plate and for the fractured tibia and modular plate ensemble are obtained.

Ribo-attenuators: novel elements for reliable and modular riboswitch engineering.

PubMed

Folliard, Thomas; Mertins, Barbara; Steel, Harrison; Prescott, Thomas P; Newport, Thomas; Jones, Christopher W; Wadhams, George; Bayer, Travis; Armitage, Judith P; Papachristodoulou, Antonis; Rothschild, Lynn J

2017-07-04

Riboswitches are structural genetic regulatory elements that directly couple the sensing of small molecules to gene expression. They have considerable potential for applications throughout synthetic biology and bio-manufacturing as they are able to sense a wide range of small molecules and regulate gene expression in response. Despite over a decade of research they have yet to reach this considerable potential as they cannot yet be treated as modular components. This is due to several limitations including sensitivity to changes in genetic context, low tunability, and variability in performance. To overcome the associated difficulties with riboswitches, we have designed and introduced a novel genetic element called a ribo-attenuator in Bacteria. This genetic element allows for predictable tuning, insulation from contextual changes, and a reduction in expression variation. Ribo-attenuators allow riboswitches to be treated as truly modular and tunable components, thus increasing their reliability for a wide range of applications.

Modular arrangement of regulatory RNA elements.

PubMed

Roßmanith, Johanna; Narberhaus, Franz

2017-03-04

Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.

An NXF1 mRNA with a retained intron is expressed in hippocampal and neocortical neurons and is translated into a protein that functions as an Nxf1 cofactor.

PubMed

Li, Ying; Bor, Yeou-Cherng; Fitzgerald, Mark P; Lee, Kevin S; Rekosh, David; Hammarskjold, Marie-Louise

2016-12-01

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356-amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes. © 2016 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The nuclear OXPHOS genes in insecta: a common evolutionary origin, a common cis-regulatory motif, a common destiny for gene duplicates

PubMed Central

Porcelli, Damiano; Barsanti, Paolo; Pesole, Graziano; Caggese, Corrado

2007-01-01

Background When orthologous sequences from species distributed throughout an optimal range of divergence times are available, comparative genomics is a powerful tool to address problems such as the identification of the forces that shape gene structure during evolution, although the functional constraints involved may vary in different genes and lineages. Results We identified and annotated in the MitoComp2 dataset the orthologs of 68 nuclear genes controlling oxidative phosphorylation in 11 Drosophilidae species and in five non-Drosophilidae insects, and compared them with each other and with their counterparts in three vertebrates (Fugu rubripes, Danio rerio and Homo sapiens) and in the cnidarian Nematostella vectensis, taking into account conservation of gene structure and regulatory motifs, and preservation of gene paralogs in the genome. Comparative analysis indicates that the ancestral insect OXPHOS genes were intron rich and that extensive intron loss and lineage-specific intron gain occurred during evolution. Comparison with vertebrates and cnidarians also shows that many OXPHOS gene introns predate the cnidarian/Bilateria evolutionary split. The nuclear respiratory gene element (NRG) has played a key role in the evolution of the insect OXPHOS genes; it is constantly conserved in the OXPHOS orthologs of all the insect species examined, while their duplicates either completely lack the element or possess only relics of the motif. Conclusion Our observations reinforce the notion that the common ancestor of most animal phyla had intron-rich gene, and suggest that changes in the pattern of expression of the gene facilitate the fixation of duplications in the genome and the development of novel genetic functions. PMID:18315839

A retained intron in the 3'-UTR of Calm3 mRNA mediates its Staufen2- and activity-dependent localization to neuronal dendrites.

PubMed

Sharangdhar, Tejaswini; Sugimoto, Yoichiro; Heraud-Farlow, Jacqueline; Fernández-Moya, Sandra M; Ehses, Janina; Ruiz de Los Mozos, Igor; Ule, Jernej; Kiebler, Michael A

2017-10-01

Dendritic localization and hence local mRNA translation contributes to synaptic plasticity in neurons. Staufen2 (Stau2) is a well-known neuronal double-stranded RNA-binding protein (dsRBP) that has been implicated in dendritic mRNA localization. The specificity of Stau2 binding to its target mRNAs remains elusive. Using individual-nucleotide resolution CLIP (iCLIP), we identified significantly enriched Stau2 binding to the 3'-UTRs of 356 transcripts. In 28 (7.9%) of those, binding occurred to a retained intron in their 3'-UTR The strongest bound 3'-UTR intron was present in the longest isoform of Calmodulin 3 ( Calm3 L ) mRNA Calm3 L 3'-UTR contains six Stau2 crosslink clusters, four of which are in this retained 3'-UTR intron. The Calm3 L mRNA localized to neuronal dendrites, while lack of the 3'-UTR intron impaired its dendritic localization. Importantly, Stau2 mediates this dendritic localization via the 3'-UTR intron, without affecting its stability. Also, NMDA-mediated synaptic activity specifically promoted the dendritic mRNA localization of the Calm3 L isoform, while inhibition of synaptic activity reduced it substantially. Together, our results identify the retained intron as a critical element in recruiting Stau2, which then allows for the localization of Calm3 L mRNA to distal dendrites. © 2017 The Authors.

Modularity: An Application of General Systems Theory to Military Force Development

DTIC Science & Technology

2005-01-01

1999). Context, modularity, and the cultural constitution of development. In P. Lloyd & C. Fernyhough (Eds.), Lev Vygotsky : Critical assessments...of General Systems Theory to Military Force Development 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...Prescribed by ANSI Std Z39-18 MODULARITY: AN APPLICATION OF GENERAL SYSTEMS THEORY TO MILITARY FORCE DEVELOPMENT 279 R SEARCH MODULARITY: AN APPLICATION OF

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

PubMed

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

The chloroplast tRNALys(UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturase-related polypeptide.

PubMed

Neuhaus, H; Link, G

1987-01-01

The trnK gene endocing the tRNALys(UUU) has been located on mustard (Sinapis alba) chloroplast DNA, 263 bp upstream of the psbA gene on the same strand. The nucleotide sequence of the trnK gene and its flanking regions as well as the putative transcription start and termination sites are shown. The 5' end of the transcript lies 121 bp upstream of the 5' tRNA coding region and is preceded by procaryotic-type "-10" and "-35" sequence elements, while the 3' end maps 2.77 kb downstream to a DNA region with possible stemloop secondary structure. The anticodon loop of the tRNALys is interrupted by a 2,574 bp intron containing a long open reading frame, which codes for 524 amino acids. Based on conserved stem and loop structures, this intron has characteristic features of a class II intron. A region near the carboxyl terminus of the derived polypeptide appears structurally related to maturases.

Modularity of a leaf moth-wing pattern and a versatile characteristic of the wing-pattern ground plan

PubMed Central

2013-01-01

Background One of the most intriguing questions in evolutionary developmental biology is how an insect acquires a mimicry pattern within its body parts. A striking example of pattern mimicry is found in the pattern diversity of moth and butterfly wings, which is thought to evolve from preexisting elements illustrated by the nymphalid ground plan (NGP). Previous studies demonstrated that individuality of the NGP facilitates the decoupling of associated common elements, leading to divergence. In contrast, recent studies on the concept of modularity have argued the importance of a combination of coupling and decoupling of the constituent elements. Here, we examine the modularity of a mimicry wing pattern in a moth and explore an evolvable characteristic of the NGP. Results This study examined the wings of the noctuid moth Oraesia excavata, which closely resemble leaves with a leaf venation pattern. Based on a comparative morphological procedure, we found that this leaf pattern was formed by the NGP common elements. Using geometric morphometrics combined with network analysis, we found that each of the modules in the leaf pattern integrates the constituent components of the leaf venation pattern (i.e., the main and lateral veins). Moreover, the detected modules were established by coupling different common elements and decoupling even a single element into different modules. The modules of the O. excavata wing pattern were associated with leaf mimicry, not with the individuality of the NGP common elements. For comparison, we also investigated the modularity of a nonmimetic pattern in the noctuid moth Thyas juno. Quantitative analysis demonstrated that the modules of the T. juno wing pattern regularly corresponded to the individuality of the NGP common elements, unlike those in the O. excavata wing pattern. Conclusions This study provides the first evidence for modularity in a leaf mimicry pattern. The results suggest that the evolution of this pattern involves coupling and decoupling processes to originate these modules, free from the individuality of the NGP system. We propose that this evolution has been facilitated by a versatile characteristic of the NGP, allowing the association of freely modifiable subordinate common elements to make modules. PMID:23890367

Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells.

PubMed

Guo, Weilong; Chung, Wen-Yu; Qian, Minping; Pellegrini, Matteo; Zhang, Michael Q

2014-03-01

DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.

Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene

PubMed Central

Gonçalves, Ana; Coelho, Teresa; Melo-Pires, Manuel; Sousa, Mário

2017-01-01

A broad mutational spectrum in the dystrophin (DMD) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD. PMID:28972564

Efficiency of introns from various origins in fish cells.

PubMed

Bétancourt, O H; Attal, J; Théron, M C; Puissant, C; Houdebine, L M

1993-06-01

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

Compositions and methods for adoptive and active immunotherapy

DOEpatents

Fahmy, Tarek; Steenblock, Erin

2014-01-14

Modular aAPCs and methods of their manufacture and use are provided. The modular aAPCs are constructed from polymeric microparticles. The aAPCs include encapsulated cytokines and coupling agents which modularly couple functional elements including T cell receptor activators, co-stimulatory molecules and adhesion molecules to the particle. The ability of these aAPCs to release cytokines in a controlled manner, coupled with their modular nature and ease of ligand attachment, results in an ideal, tunable APC capable of stimulating and expanding primary T cells.

BeamDyn: a high-fidelity wind turbine blade solver in the FAST modular framework

DOE PAGES

Wang, Qi; Sprague, Michael A.; Jonkman, Jason; ...

2017-03-14

Here, this paper presents a numerical implementation of the geometrically exact beam theory based on the Legendre-spectral-finite-element (LSFE) method. The displacement-based geometrically exact beam theory is presented, and the special treatment of three-dimensional rotation parameters is reviewed. An LSFE is a high-order finite element with nodes located at the Gauss-Legendre-Lobatto points. These elements can be an order of magnitude more computationally efficient than low-order finite elements for a given accuracy level. The new module, BeamDyn, is implemented in the FAST modularization framework for dynamic simulation of highly flexible composite-material wind turbine blades within the FAST aeroelastic engineering model. The frameworkmore » allows for fully interactive simulations of turbine blades in operating conditions. Numerical examples are provided to validate BeamDyn and examine the LSFE performance as well as the coupling algorithm in the FAST modularization framework. BeamDyn can also be used as a stand-alone high-fidelity beam tool.« less

BeamDyn: a high-fidelity wind turbine blade solver in the FAST modular framework

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

Wang, Qi; Sprague, Michael A.; Jonkman, Jason

Here, this paper presents a numerical implementation of the geometrically exact beam theory based on the Legendre-spectral-finite-element (LSFE) method. The displacement-based geometrically exact beam theory is presented, and the special treatment of three-dimensional rotation parameters is reviewed. An LSFE is a high-order finite element with nodes located at the Gauss-Legendre-Lobatto points. These elements can be an order of magnitude more computationally efficient than low-order finite elements for a given accuracy level. The new module, BeamDyn, is implemented in the FAST modularization framework for dynamic simulation of highly flexible composite-material wind turbine blades within the FAST aeroelastic engineering model. The frameworkmore » allows for fully interactive simulations of turbine blades in operating conditions. Numerical examples are provided to validate BeamDyn and examine the LSFE performance as well as the coupling algorithm in the FAST modularization framework. BeamDyn can also be used as a stand-alone high-fidelity beam tool.« less

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

PubMed Central

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

1995-01-01

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

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence.

PubMed

Shin, Dong-Ho; Webb, Barbara M; Nakao, Miki; Smith, Sylvia L

2009-07-01

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence

PubMed Central

Shin, Dong-Ho; Webb, Barbara M.; Nakao, Miki; Smith, Sylvia L.

2009-01-01

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and –d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (≤) amino acid identities with each other, 35.4 ~ 39.6% and 62.8 ~ 65.9% with factor I of mammals and banded houndshark (Triakis scyllium), respectively. The modular structure of the GcIf is similar to that of mammals with one notable exception, the presence of a novel shark-specific sequence between the leader peptide (LP) and the factor I membrane attack complex (FIMAC) domain. The cDNA sequences differ only in the size and composition of the shark-specific region (SSR). Sequence analysis of each SSR has identified within the region two novel short sequences (SS1 and SS2) and three repeat sequences (RS1, 2 and 3). Genomic analysis has revealed the existence of three introns between the leader peptide and the FIMAC domain, tentatively designated intron 1, intron 2, and intron 3 which span 4067, 2293 and 2082 bp, respectively. Southern blot analysis suggests the presence of a single gene copy for each cDNA type. Phylogenetic analysis suggests that complement factor I of cartilaginous fish diverged prior to the emergence of mammals. All four GcIf cDNA species are expressed in four different tissues and the liver is the main tissue in which expression level of all four is high. This suggests that the expression of GcIf isotypes is tissue-dependent. PMID:19423168

A var gene promoter implicated in severe malaria nucleates silencing and is regulated by 3’ untranslated region and intronic cis-elements

PubMed Central

Muhle, Rebecca A.; Adjalley, Sophie; Falkard, Brie; Nkrumah, Louis J.; Muhle, Michael E.; Fidock, David A.

2009-01-01

Questions surround the mechanism of mutually exclusive expression by which Plasmodium falciparum mediates activation and silencing of var genes. These encode PfEMP1 proteins, which function as cytoadherent and immunomodulatory molecules at the surface of parasitized erythrocytes. Current evidence suggests that promoter silencing by var introns might play a key role in var gene regulation. To evaluate the impact of cis-acting regulatory regions on var silencing, we generated P. falciparum lines in which luciferase was placed under the control of an UpsA var promoter. By utilizing the Bxb1 integrase system, these reporter cassettes were targeted to a genomic region that was not in apposition to var sub-telomeric domains. This eliminated possible effects from surrounding telomeric elements and removed the variability inherent in episomal systems. Studies with highly synchronized parasites revealed that the UpsA element possessed minimal activity in comparison with a heterologous (hrp3) promoter. This may well result from the integrated UpsA promoter being largely silenced by the neighboring cg6 promoter. Our analyses also revealed that the DownsA 3’ untranslated region further decreased the luciferase activity from both cassettes, whereas the var A intron repressed the UpsA promoter specifically. By applying multivariate analysis over the entire cell cycle, we confirmed the significance of these cis-elements and found the parasite stage to be the major factor regulating UpsA promoter activity. Additionally, we observed that the UpsA promoter was capable of nucleating reversible silencing that spread to a downstream promoter. We believe these studies are the first to analyze promoter activity of Group A var genes which have been implicated in severe malaria, and support the model that var introns can further suppress var expression. These data also suggest an important suppressive role for the DownsA terminator. Our findings imply the existence of multiple levels of var gene regulation in addition to intrinsic promoter-dependent silencing. PMID:19463825

Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements

PubMed Central

Gogarten, J Peter; Hilario, Elena

2006-01-01

Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer) than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6:39) and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6:42) provide important stepping stones towards integrated studies on how these parasitic elements evolve through time together with, or despite, their hosts. PMID:17101053

Design of a modular digital computer system, CDRL no. D001, final design plan

NASA Technical Reports Server (NTRS)

Easton, R. A.

1975-01-01

The engineering breadboard implementation for the CDRL no. D001 modular digital computer system developed during design of the logic system was documented. This effort followed the architecture study completed and documented previously, and was intended to verify the concepts of a fault tolerant, automatically reconfigurable, modular version of the computer system conceived during the architecture study. The system has a microprogrammed 32 bit word length, general register architecture and an instruction set consisting of a subset of the IBM System 360 instruction set plus additional fault tolerance firmware. The following areas were covered: breadboard packaging, central control element, central processing element, memory, input/output processor, and maintenance/status panel and electronics.

The paradox of MHC-DRB exon/intron evolution: alpha-helix and beta-sheet encoding regions diverge while hypervariable intronic simple repeats coevolve with beta-sheet codons.

PubMed

Schwaiger, F W; Weyers, E; Epplen, C; Brün, J; Ruff, G; Crawford, A; Epplen, J T

1993-09-01

Twenty-one different caprine and 13 ovine MHC-DRB exon 2 sequences were determined including part of the adjacent introns containing simple repetitive (gt)n(ga)m elements. The positions for highly polymorphic DRB amino acids vary slightly among ungulates and other mammals. From man and mouse to ungulates the basic (gt)n(ga)m structure is fixed in evolution for 7 x 10(7) years whereas ample variations exist in the tandem (gt)n and (ga)m dinucleotides and especially their "degenerated" derivatives. Phylogenetic trees for the alpha-helices and beta-pleated sheets of the ungulate DRB sequences suggest different evolutionary histories. In hoofed animals as well as in humans DRB beta-sheet encoding sequences and adjacent intronic repeats can be assembled into virtually identical groups suggesting coevolution of noncoding as well as coding DNA. In contrast alpha-helices and C-terminal parts of the first DRB domain evolve distinctly. In the absence of a defined mechanism causing specific, site-directed mutations, double-recombination or gene-conversion-like events would readily explain this fact. The role of the intronic simple (gt)n(ga)m repeat is discussed with respect to these genetic exchange mechanisms during evolution.

Antisense Masking of an hnRNP A1/A2 Intronic Splicing Silencer Corrects SMN2 Splicing in Transgenic Mice

PubMed Central

Hua, Yimin; Vickers, Timothy A.; Okunola, Hazeem L.; Bennett, C. Frank; Krainer, Adrian R.

2008-01-01

survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA. PMID:18371932

Potential Applications of Modularity to Enable a Deep Space Habitation Capability for Future Human Exploration Beyond Low-Earth Orbit

NASA Technical Reports Server (NTRS)

Simon, Matthew A.; Toups, Larry; Smitherman, David

2012-01-01

Evaluating preliminary concepts of a Deep Space Habitat (DSH) enabling long duration crewed exploration of asteroids, the Moon, and Mars is a technically challenging problem. Sufficient habitat volumes and equipment, necessary to ensure crew health and functionality, increase propellant requirements and decrease launch flexibility to deliver multiple elements on a single launch vehicle; both of which increase overall mission cost. Applying modularity in the design of the habitat structures and subsystems can alleviate these difficulties by spreading the build-up of the overall habitation capability across several smaller parts. This allows for a more flexible habitation approach that accommodates various crew mission durations and levels of functionality. This paper provides a technical analysis of how various modular habitation approaches can impact the parametric design of a DSH with potential benefits in mass, packaging volume, and architectural flexibility. This includes a description of the desired long duration habitation capability, the definition of a baseline model for comparison, a small trade study to investigate alternatives, and commentary on potentially advantageous configurations to enable different levels of habitability. The approaches investigated include modular pressure vessel strategies, modular subsystems, and modular manufacturing approaches to habitat structure. The paper also comments upon the possibility of an integrated habitation strategy using modular components to create all short and long duration habitation elements required in the current exploration architectures.

A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification.

PubMed

Cenik, Can; Chua, Hon Nian; Singh, Guramrit; Akef, Abdalla; Snyder, Michael P; Palazzo, Alexander F; Moore, Melissa J; Roth, Frederick P

2017-03-01

Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5 ' proximal- i ntron- m inus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the exon junction complex (EJC) at noncanonical 5' proximal positions. Finally, N 1 -methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising ∼20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N 1 -methyladenosines in the early coding region, and enrichment for noncanonical binding by the EJC. © 2017 Cenik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Identification of Genetic Elements Associated with EPSPS Gene Amplification

PubMed Central

Gaines, Todd A.; Wright, Alice A.; Molin, William T.; Lorentz, Lothar; Riggins, Chance W.; Tranel, Patrick J.; Beffa, Roland; Westra, Philip; Powles, Stephen B.

2013-01-01

Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world’s most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S) A. palmeri, and that only one of these was amplified in glyphosate-resistant (R) A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs) were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac) transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene. PMID:23762434

CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.

PubMed

Bryant, Jessica M; Regnault, Clément; Scheidig-Benatar, Christine; Baumgarten, Sebastian; Guizetti, Julien; Scherf, Artur

2017-07-11

Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression. Copyright © 2017 Bryant et al.

Automatic Assembly of Combined Checkingfixture for Auto-Body Components Based Onfixture Elements Libraries

NASA Astrophysics Data System (ADS)

Jiang, Jingtao; Sui, Rendong; Shi, Yan; Li, Furong; Hu, Caiqi

In this paper 3-D models of combined fixture elements are designed, classified by their functions, and saved in computer as supporting elements library, jointing elements library, basic elements library, localization elements library, clamping elements library, and adjusting elements library etc. Then automatic assembly of 3-D combined checking fixture for auto-body part is presented based on modularization theory. And in virtual auto-body assembly space, Locating constraint mapping technique and assembly rule-based reasoning technique are used to calculate the position of modular elements according to localization points and clamp points of auto-body part. Auto-body part model is transformed from itself coordinate system space to virtual assembly space by homogeneous transformation matrix. Automatic assembly of different functional fixture elements and auto-body part is implemented with API function based on the second development of UG. It is proven in practice that the method in this paper is feasible and high efficiency.

Pseudoexon activation increases phenotype severity in a Becker muscular dystrophy patient.

PubMed

Greer, Kane; Mizzi, Kayla; Rice, Emily; Kuster, Lukas; Barrero, Roberto A; Bellgard, Matthew I; Lynch, Bryan J; Foley, Aileen Reghan; O Rathallaigh, Eoin; Wilton, Steve D; Fletcher, Sue

2015-07-01

We report a dystrophinopathy patient with an in-frame deletion of DMD exons 45-47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.

Towards a Formal Basis for Modular Safety Cases

NASA Technical Reports Server (NTRS)

Denney, Ewen; Pai, Ganesh

2015-01-01

Safety assurance using argument-based safety cases is an accepted best-practice in many safety-critical sectors. Goal Structuring Notation (GSN), which is widely used for presenting safety arguments graphically, provides a notion of modular arguments to support the goal of incremental certification. Despite the efforts at standardization, GSN remains an informal notation whereas the GSN standard contains appreciable ambiguity especially concerning modular extensions. This, in turn, presents challenges when developing tools and methods to intelligently manipulate modular GSN arguments. This paper develops the elements of a theory of modular safety cases, leveraging our previous work on formalizing GSN arguments. Using example argument structures we highlight some ambiguities arising through the existing guidance, present the intuition underlying the theory, clarify syntax, and address modular arguments, contracts, well-formedness and well-scopedness of modules. Based on this theory, we have a preliminary implementation of modular arguments in our toolset, AdvoCATE.

Clinical Physiologic Research Instrumentation: An Approach Using Modular Elements and Distributed Processing

PubMed Central

Hagen, R. W.; Ambos, H. D.; Browder, M. W.; Roloff, W. R.; Thomas, L. J.

1979-01-01

The Clinical Physiologic Research System (CPRS) developed from our experience in applying computers to medical instrumentation problems. This experience revealed a set of applications with a commonality in data acquisition, analysis, input/output, and control needs that could be met by a portable system. The CPRS demonstrates a practical methodology for integrating commercial instruments with distributed modular elements of local design in order to make facile responses to changing instrumentation needs in clinical environments. ImagesFigure 3

A KCNH2 branch point mutation causing aberrant splicing contributes to an explanation of genotype-negative long QT syndrome.

PubMed

Crotti, Lia; Lewandowska, Marzena A; Schwartz, Peter J; Insolia, Roberto; Pedrazzini, Matteo; Bussani, Erica; Dagradi, Federica; George, Alfred L; Pagani, Franco

2009-02-01

Genetic screening of long QT syndrome (LQTS) fails to identify disease-causing mutations in about 30% of patients. So far, molecular screening has focused mainly on coding sequence mutations or on substitutions at canonical splice sites. The purpose of this study was to explore the possibility that intronic variants not at canonical splice sites might affect splicing regulatory elements, lead to aberrant transcripts, and cause LQTS. Molecular screening was performed through DHPLC and sequence analysis. The role of the intronic mutation identified was assessed with a hybrid minigene splicing assay. A three-generation LQTS family was investigated. Molecular screening failed to identify an obvious disease-causing mutation in the coding sequences of the major LQTS genes but revealed an intronic A-to-G substitution in KCNH2 (IVS9-28A/G) cosegregating with the clinical phenotype in family members. In vitro analysis proved that the mutation disrupts the acceptor splice site definition by affecting the branch point (BP) sequence and promoting intron retention. We further demonstrated a tight functional relationship between the BP and the polypyrimidine tract, whose weakness is responsible for the pathological effect of the IVS9-28A/G mutation. We identified a novel BP mutation in KCNH2 that disrupts the intron 9 acceptor splice site definition and causes LQT2. The present finding demonstrates that intronic mutations affecting pre-mRNA processing may contribute to the failure of traditional molecular screening in identifying disease-causing mutations in LQTS subjects and offers a rationale strategy for the reduction of genotype-negative cases.

A Modular Robotic System with Applications to Space Exploration

NASA Technical Reports Server (NTRS)

Hancher, Matthew D.; Hornby, Gregory S.

2006-01-01

Modular robotic systems offer potential advantages as versatile, fault-tolerant, cost-effective platforms for space exploration, but a sufficiently mature system is not yet available. We describe the possible applications of such a system, and present prototype hardware intended as a step in the right direction. We also present elements of an automated design and optimization framework aimed at making modular robots easier to design and use, and discuss the results of applying the system to a gait optimization problem. Finally, we discuss the potential near-term applications of modular robotics to terrestrial robotics research.

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription.

PubMed

Akay, Alper; Di Domenico, Tomas; Suen, Kin M; Nabih, Amena; Parada, Guillermo E; Larance, Mark; Medhi, Ragini; Berkyurek, Ahmet C; Zhang, Xinlian; Wedeles, Christopher J; Rudolph, Konrad L M; Engelhardt, Jan; Hemberg, Martin; Ma, Ping; Lamond, Angus I; Claycomb, Julie M; Miska, Eric A

2017-08-07

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.

PubMed Central

Scieglinska, D; Widłak, W; Konopka, W; Poutanen, M; Rahman, N; Huhtaniemi, I; Krawczyk, Z

2001-01-01

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences. PMID:11563976

Cis-regulatory underpinnings of human GLI3 expression in embryonic craniofacial structures and internal organs.

PubMed

Abbasi, Amir A; Minhas, Rashid; Schmidt, Ansgar; Koch, Sabine; Grzeschik, Karl-Heinz

2013-10-01

The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Short intronic repeat sequences facilitate circular RNA production

PubMed Central

Liang, Dongming

2014-01-01

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery “backsplices” and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. PMID:25281217

The endogenous transposable element Tgm9 is suitable for functional analyses of soybean genes and generating novel mutants for genetic improvement of soybean

USDA-ARS?s Scientific Manuscript database

In soybean, variegated flowers can be caused by somatic excision of the CACTA-type transposable element Tgm9 from intron 2 of the DFR2 gene encoding dihydroflavonol-4-reductase in the anthocyanin pigment biosynthetic pathway. DFR2 has been mapped to the W4 locus where the allele containing the elem...

Design of a modular digital computer system

NASA Technical Reports Server (NTRS)

1980-01-01

A Central Control Element (CCE) module which controls the Automatically Reconfigurable Modular System (ARMS) and allows both redundant processing and multi-computing in the same computer with real time mode switching, is discussed. The same hardware is used for either reliability enhancement, speed enhancement, or for a combination of both.

Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle.

PubMed

Anggraeni, Melisa R; Connors, Natalie K; Wu, Yang; Chuan, Yap P; Lua, Linda H L; Middelberg, Anton P J

2013-09-13

Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Simulation of Thermal Behavior in High-Precision Measurement Instruments

NASA Astrophysics Data System (ADS)

Weis, Hanna Sophie; Augustin, Silke

2008-06-01

In this paper, a way to modularize complex finite-element models is described. The modularization is done with temperature fields that appear in high-precision measurement instruments. There, the temperature negatively impacts the achievable uncertainty of measurement. To correct for this uncertainty, the temperature must be known at every point. This cannot be achieved just by measuring temperatures at specific locations. Therefore, a numerical treatment is necessary. As the system of interest is very complex, modularization is unavoidable to obtain good numerical results.

I(CES)-cubes: a modular self-reconfigurable bipartite robotic system

NASA Astrophysics Data System (ADS)

Unsal, Cem; Kiliccote, Han; Khosla, Pradeep K.

1999-08-01

In this manuscript, we introduce I(CES)-Cubes, a class of 3D modular robotic system that is capable of reconfiguring itself in order to adapt to its environment. This is a bipartite system, i.e. a collection of (i) active elements capable of actuation, and (ii) passive elements acting as connectors between actuated elements. Active elements, called links, are 3-DOF manipulators that are capable of attaching/detaching themselves to/from the passive elements. The cubes can then be positioned and oriented using links, which are independent mechatronic elements. Self- reconfiguration property enables the system to performed locomotion tasks over difficult terrain. For example, the system would be capable of moving over obstacles and climbing stairs. These task are performed by positing and orienting cubes and links to form a 3D network with required shape and position. This paper describes the design of the passive and active elements, the attachment mechanics, and several reconfiguration scenarios. Specifics of the hardware implementation and result of experiments with current prototypes are also given.

Construction concepts and validation of the 3D printed UST_2 modular stellarator

NASA Astrophysics Data System (ADS)

Queral, V.

2015-03-01

High accuracy, geometric complexity and thus high cost of stellarators tend to hinder the advance of stellarator research. Nowadays, new manufacturing methods might be developed for the production of small and middle-size stellarators. The methods should demonstrate advantages with respect common fabrication methods, like casting, cutting, forging and welding, for the construction of advanced highly convoluted modular stellarators. UST2 is a small modular three period quasi-isodynamic stellarator of major radius 0.26 m and plasma volume 10 litres being currently built to validate additive manufacturing (3D printing) for stellarator construction. The modular coils are wound in grooves defined on six 3D printed half period frames designed as light truss structures filled by a strong filler. A geometrically simple assembling configuration has been concocted for UST2 so as to try to lower the cost of the device while keeping the positioning accuracy of the different elements. The paper summarizes the construction and assembling concepts developed, the devised positioning methodology, the design of the coil frames and positioning elements and, an initial validation of the assembling of the components.

Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes

PubMed Central

Adir, Idan; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

2016-01-01

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal’s lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene’s promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species. PMID:27560950

Porcine calbindin-D9k gene: expression in endometrium, myometrium, and placenta in the absence of a functional estrogen response element in intron A.

PubMed

Krisinger, J; Jeung, E B; Simmen, R C; Leung, P C

1995-01-01

The expression of Calbindin-D9k (CaBP-9k) in the pig uterus and placenta was measured by Northern blot analysis and reverse transcription polymerase chain reaction (PCR), respectively. Progesterone (P4) administration to ovariectomized pigs decreased CaBP-9k mRNA levels. Expression of endometrial CaBP-9k mRNA was high on pregnancy Days 10-12 and below the detection limit on Days 15 and 18. On Day 60, expression could be detected at low levels. In myometrium and placenta, CaBP-9k mRNA expression was not detectable by Northern analysis using total RNA. Reverse-transcribed RNA from both tissues demonstrated the presence of CaBP-9k transcripts by means of PCR. The partial CaBP-9k gene was amplified by PCR and cloned to determine the sequence of intron A. In contrast to the rat CaBP-9k gene, the pig gene does not contain a functional estrogen response element (ERE) within this region. A similar ERE-like sequence located at the identical location was examined by gel retardation analysis and failed to bind the estradiol receptor. A similar disruption of this ERE-like sequence has been described in the human CaBP-9k gene, which is not expressed at any level in placenta, myometrium, or endometrium. It is concluded that the pig CaBP-9k gene is regulated in these reproductive tissues in a manner distinct from that in rat and human tissues. The regulation is probably due to a regulatory region outside of intron A, which in the rat gene contains the key cis element for uterine expression of the CaBP-9k gene.

Co-regulation of nuclear respiratory factor-1 by NFκB and CREB links LPS-induced inflammation to mitochondrial biogenesis

PubMed Central

Suliman, Hagir B.; Sweeney, Timothy E.; Withers, Crystal M.; Piantadosi, Claude A.

2010-01-01

The nuclear respiratory factor-1 (NRF1) gene is activated by lipopolysaccharide (LPS), which might reflect TLR4-mediated mitigation of cellular inflammatory damage via initiation of mitochondrial biogenesis. To test this hypothesis, we examined NRF1 promoter regulation by NFκB, and identified interspecies-conserved κB-responsive promoter and intronic elements in the NRF1 locus. In mice, activation of Nrf1 and its downstream target, Tfam, by Escherichia coli was contingent on NFκB, and in LPS-treated hepatocytes, NFκB served as an NRF1 enhancer element in conjunction with NFκB promoter binding. Unexpectedly, optimal NRF1 promoter activity after LPS also required binding by the energy-state-dependent transcription factor CREB. EMSA and ChIP assays confirmed p65 and CREB binding to the NRF1 promoter and p65 binding to intron 1. Functionality for both transcription factors was validated by gene-knockdown studies. LPS regulation of NRF1 led to mtDNA-encoded gene expression and expansion of mtDNA copy number. In cells expressing plasmid constructs containing the NRF-1 promoter and GFP, LPS-dependent reporter activity was abolished by cis-acting κB-element mutations, and nuclear accumulation of NFκB and CREB demonstrated dependence on mitochondrial H2O2. These findings indicate that TLR4-dependent NFκB and CREB activation co-regulate the NRF1 promoter with NFκB intronic enhancement and redox-regulated nuclear translocation, leading to downstream target-gene expression, and identify NRF-1 as an early-phase component of the host antibacterial defenses. PMID:20587593

Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

PubMed

Miyata, A; Yokoyama, C; Ihara, H; Bandoh, S; Takeda, O; Takahashi, E; Tanabe, T

1994-09-01

The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

An out-of-core thermionic-converter system for nuclear space power

NASA Technical Reports Server (NTRS)

Breitwieser, R.

1972-01-01

Design of the nuclear thermionic space power system, 40 50 70 Kw(e) power range, are given. The design configuration (1) meets the constraints of readily available launch vehicles; (2) allows for off-design operation including startup, shutdown, and possible emergency conditions; (3) provides tolerance of failure by extensive use of modular, redundant elements; (4) incorporates and uses heat pipes in a fashion that reduces the need for extensive in-pile testing of system components; and (5) uses thermionic converters, nuclear fuel elements, and heat transfer devices in a geometrical form adapted from existing incore thermionic system designs. Designs and in some cases performance data for elements and groups of the elements of the system are included. Benefits of the highly modular system approach to reliability, safety, economy of development, and flexibility are discussed.

Short intronic repeat sequences facilitate circular RNA production.

PubMed

Liang, Dongming; Wilusz, Jeremy E

2014-10-15

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. © 2014 Liang and Wilusz; Published by Cold Spring Harbor Laboratory Press.

Genome Analysis Reveals Interplay between 5′UTR Introns and Nuclear mRNA Export for Secretory and Mitochondrial Genes

PubMed Central

Cenik, Can; Chua, Hon Nian; Zhang, Hui; Tarnawsky, Stefan P.; Akef, Abdalla; Derti, Adnan; Tasan, Murat; Moore, Melissa J.; Palazzo, Alexander F.; Roth, Frederick P.

2011-01-01

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5′ end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR–containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX–promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5′ untranslated region (5′UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export. PMID:21533221

Splicing predictions reliably classify different types of alternative splicing

PubMed Central

Busch, Anke; Hertel, Klemens J.

2015-01-01

Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5′ or 3′ splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements. PMID:25805853

The type I fatty acid and polyketide synthases: a tale of two megasynthases

PubMed Central

Tsai, Shiou-Chuan

2008-01-01

This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

Human life support during interplanetary travel and domicile. II - Generic Modular Flow Schematic modeling

NASA Technical Reports Server (NTRS)

Farral, Joseph F.; Seshan, P. K.; Rohatgi, Naresh K.

1991-01-01

This paper describes the Generic Modular Flow Schematic (GMFS) architecture capable of encompassing all functional elements of a physical/chemical life support system (LSS). The GMFS can be implemented to synthesize, model, analyze, and quantitatively compare many configurations of LSSs, from a simple, completely open-loop to a very complex closed-loop. The GMFS model is coded in ASPEN, a state-of-the-art chemical process simulation program, to accurately compute the material, heat, and power flow quantities for every stream in each of the subsystem functional elements (SFEs) in the chosen configuration of a life support system. The GMFS approach integrates the various SFEs and subsystems in a hierarchical and modular fashion facilitating rapid substitutions and reconfiguration of a life support system. The comprehensive ASPEN material and energy balance output is transferred to a systems and technology assessment spreadsheet for rigorous system analysis and trade studies.

Modularity and hierarchical organization of action programs in children's acquisition of graphic skills.

PubMed

Manoel, Edison de J; Dantas, Luiz; Gimenez, Roberto; de Oliveira, Dalton Lustosa

2011-10-01

The organization of actions is based on modules in memory as a result of practice, easing the demand of performing more complex actions. If this modularization occurs, the elements of the module must remain invariant in new tasks. To test this hypothesis, 35 children, age 10 yr., practiced a graphic criterion task on a digital tablet and completed a complex graphic task enclosing the previous one. Total movement and pause times to draw the figure indicated skill acquisition. A module was identified by the variability of relative timing, pause time, and sequencing. Total movement to perform the criterion task did not increase significantly when it was embedded in the more complex task. Modularity was evidenced by the stability of relative timing and pause time and sequencing. The spatial position of new elements did not perturb the module, so the grammar of action may still have been forming.

Modular architecture for robotics and teleoperation

DOEpatents

Anderson, Robert J.

1996-12-03

Systems and methods for modularization and discretization of real-time robot, telerobot and teleoperation systems using passive, network based control laws. Modules consist of network one-ports and two-ports. Wave variables and position information are passed between modules. The behavior of each module is decomposed into uncoupled linear-time-invariant, and coupled, nonlinear memoryless elements and then are separately discretized.

Alu distribution and mutation types of cancer genes

PubMed Central

2011-01-01

Background Alu elements are the most abundant retrotransposable elements comprising ~11% of the human genome. Many studies have highlighted the role that Alu elements have in genetic instability and how their contribution to the assortment of mutagenic events can lead to cancer. As of yet, little has been done to quantitatively assess the association between Alu distribution and genes that are causally implicated in oncogenesis. Results We have investigated the effect of various Alu densities on the mutation type based classifications of cancer genes. In order to establish the direct relationship between Alus and the cancer genes of interest, genome wide Alu-related densities were measured using genes rather than the sliding windows of fixed length as the units. Several novel genomic features, such as the density of the adjacent Alu pairs and the number of Alu-Exon-Alu triplets, were developed in order to extend the investigation via the multivariate statistical analysis toward more advanced biological insight. In addition, we characterized the genome-wide intron Alu distribution with a mixture model that distinguished genes containing Alu elements from those with no Alus, and evaluated the gene-level effect of the 5'-TTAAAA motif associated with Alu insertion sites using a two-step regression analysis method. Conclusions The study resulted in several novel findings worthy of further investigation. They include: (1) Recessive cancer genes (tumor suppressor genes) are enriched with Alu elements (p < 0.01) compared to dominant cancer genes (oncogenes) and the entire set of genes in the human genome; (2) Alu-related genomic features can be used to cluster cancer genes into biological meaningful groups; (3) The retention of exon Alus has been restricted in the human genome development, and an upper limit to the chromosome-level exon Alu densities is suggested by the distribution profile; (4) For the genes with at least one intron Alu repeat in individual chromosomes, the intron Alu densities can be well fitted by a Gamma distribution; (5) The effect of the 5'-TTAAAA motif on Alu densities varies across different chromosomes. PMID:21429208

Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

PubMed

Sam, Mohammad Reza; Azadbakhsh, Azadeh Sadat; Farokhi, Farrah; Rezazadeh, Kobra; Sam, Sohrab; Zomorodipour, Alireza; Haddad-Mashadrizeh, Aliakbar; Delirezh, Nowruz; Mokarizadeh, Aram

2016-05-01

Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Hardening Effect Analysis by Modular Upper Bound and Finite Element Methods in Indentation of Aluminum, Steel, Titanium and Superalloys

PubMed Central

Bermudo, Carolina; Sevilla, Lorenzo; Martín, Francisco; Trujillo, Francisco Javier

2017-01-01

The application of incremental processes in the manufacturing industry is having a great development in recent years. The first stage of an Incremental Forming Process can be defined as an indentation. Because of this, the indentation process is starting to be widely studied, not only as a hardening test but also as a forming process. Thus, in this work, an analysis of the indentation process under the new Modular Upper Bound perspective has been performed. The modular implementation has several advantages, including the possibility of the introduction of different parameters to extend the study, such as the friction effect, the temperature or the hardening effect studied in this paper. The main objective of the present work is to analyze the three hardening models developed depending on the material characteristics. In order to support the validation of the hardening models, finite element analyses of diverse materials under an indentation are carried out. Results obtained from the Modular Upper Bound are in concordance with the results obtained from the numerical analyses. In addition, the numerical and analytical methods are in concordance with the results previously obtained in the experimental indentation of annealed aluminum A92030. Due to the introduction of the hardening factor, the new modular distribution is a suitable option for the analysis of indentation process. PMID:28772914

The alternative oxidase family of Vitis vinifera reveals an attractive model to study the importance of genomic design.

PubMed

Costa, José Hélio; de Melo, Dirce Fernandes; Gouveia, Zélia; Cardoso, Hélia Guerra; Peixe, Augusto; Arnholdt-Schmitt, Birgit

2009-12-01

'Genomic design' refers to the structural organization of gene sequences. Recently, the role of intron sequences for gene regulation is being better understood. Further, introns possess high rates of polymorphism that are considered as the major source for speciation. In molecular breeding, the length of gene-specific introns is recognized as a tool to discriminate genotypes with diverse traits of agronomic interest. 'Economy selection' and 'time-economy selection' have been proposed as models for explaining why highly expressed genes typically contain small introns. However, in contrast to these theories, plant-specific selection reveals that highly expressed genes contain introns that are large. In the presented research, 'wet'Aox gene identification from grapevine is advanced by a bioinformatics approach to study the species-specific organization of Aox gene structures in relation to available expressed sequence tag (EST) data. Two Aox1 and one Aox2 gene sequences have been identified in Vitis vinifera using grapevine cultivars from Portugal and Germany. Searching the complete genome sequence data of two grapevine cultivars confirmed that V. vinifera alternative oxidase (Aox) is encoded by a small multigene family composed of Aox1a, Aox1b and Aox2. An analysis of EST distribution revealed high expression of the VvAox2 gene. A relationship between the atypical long primary transcript of VvAox2 (in comparison to other plant Aox genes) and its expression level is suggested. V. vinifera Aox genes contain four exons interrupted by three introns except for Aox1a which contains an additional intron in the 3'-UTR. The lengths of primary Aox transcripts were estimated for each gene in two V. vinifera varieties: PN40024 and Pinot Noir. In both varieties, Aox1a and Aox1b contained small introns that corresponded to primary transcript lengths ranging from 1501 to 1810 bp. The Aox2 of PN40024 (12 329 bp) was longer than that from Pinot Noir (7279 bp) because of selection against a transposable-element insertion that is 5028 bp in size. An EST database basic local alignment search tool (BLAST) search of GenBank revealed the following ESTs percentages for each gene: Aox1a (26.2%), Aox1b (11.9%) and Aox2 (61.9%). Aox1a was expressed in fruits and roots, Aox1b expression was confined to flowers and Aox2 was ubiquitously expressed. These data for V. vinifera show that atypically long Aox intron lengths are related to high levels of gene expression. Furthermore, it is shown for the first time that two grapevine cultivars can be distinguished by Aox intron length polymorphism.

A Best Practice Modular Design of a Hybrid Course Delivery Structure for an Executive Education Program

ERIC Educational Resources Information Center

Klotz, Dorothy E.; Wright, Thomas A.

2017-01-01

This article highlights a best practice approach that showcases the highly successful deployment of a hybrid course delivery structure for an Operations core course in an Executive MBA Program. A key design element of the approach was the modular design of both the course itself and the learning materials. While other hybrid deployments may stress…

Phase distribution of spliceosomal introns: implications for intron origin

PubMed Central

Nguyen, Hung D; Yoshihama, Maki; Kenmochi, Naoya

2006-01-01

Background The origin of spliceosomal introns is the central subject of the introns-early versus introns-late debate. The distribution of intron phases is non-uniform, with an excess of phase-0 introns. Introns-early explains this by speculating that a fraction of present-day introns were present between minigenes in the progenote and therefore must lie in phase-0. In contrast, introns-late predicts that the nonuniformity of intron phase distribution reflects the nonrandomness of intron insertions. Results In this paper, we tested the two theories using analyses of intron phase distribution. We inferred the evolution of intron phase distribution from a dataset of 684 gene orthologs from seven eukaryotes using a maximum likelihood method. We also tested whether the observed intron phase distributions from 10 eukaryotes can be explained by intron insertions on a genome-wide scale. In contrast to the prediction of introns-early, the inferred evolution of intron phase distribution showed that the proportion of phase-0 introns increased over evolution. Consistent with introns-late, the observed intron phase distributions matched those predicted by an intron insertion model quite well. Conclusion Our results strongly support the introns-late hypothesis of the origin of spliceosomal introns. PMID:16959043

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6.

PubMed

Ochiai, Kyoko; Muto, Akihiko; Tanaka, Hiromu; Takahashi, Shinichiro; Igarashi, Kazuhiko

2008-03-01

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.

Molecular Evolution of the Non-Coding Eosinophil Granule Ontogeny Transcript

PubMed Central

Rose, Dominic; Stadler, Peter F.

2011-01-01

Eukaryotic genomes are pervasively transcribed. A large fraction of the transcriptional output consists of long, mRNA-like, non-protein-coding transcripts (mlncRNAs). The evolutionary history of mlncRNAs is still largely uncharted territory. In this contribution, we explore in detail the evolutionary traces of the eosinophil granule ontogeny transcript (EGOT), an experimentally confirmed representative of an abundant class of totally intronic non-coding transcripts (TINs). EGOT is located antisense to an intron of the ITPR1 gene. We computationally identify putative EGOT orthologs in the genomes of 32 different amniotes, including orthologs from primates, rodents, ungulates, carnivores, afrotherians, and xenarthrans, as well as putative candidates from basal amniotes, such as opossum or platypus. We investigate the EGOT gene phylogeny, analyze patterns of sequence conservation, and the evolutionary conservation of the EGOT gene structure. We show that EGO-B, the spliced isoform, may be present throughout the placental mammals, but most likely dates back even further. We demonstrate here for the first time that the whole EGOT locus is highly structured, containing several evolutionary conserved, and thermodynamic stable secondary structures. Our analyses allow us to postulate novel functional roles of a hitherto poorly understood region at the intron of EGO-B which is highly conserved at the sequence level. The region contains a novel ITPR1 exon and also conserved RNA secondary structures together with a conserved TATA-like element, which putatively acts as a promoter of an independent regulatory element. PMID:22303364

Regulation of expression of transgenes in developing fish.

PubMed

Moav, B; Liu, Z; Caldovic, L D; Gross, M L; Faras, A J; Hackett, P B

1993-05-01

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

PubMed Central

Binder, Andreas; Lambert, Jayne; Morbitzer, Robert; Popp, Claudia; Ott, Thomas; Lahaye, Thomas; Parniske, Martin

2014-01-01

The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct. PMID:24551083

Identification of cis-Acting Elements and Splicing Factors Involved in the Regulation of BIM Pre-mRNA Splicing

PubMed Central

Juan, Wen Chun; Roca, Xavier; Ong, S. Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3′ end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes. PMID:24743263

Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

PubMed

Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes

PubMed Central

López-Garriga, Juan; Cadilla, Carmen L.

2016-01-01

The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5’end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments. PMID:26824233

Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene.

PubMed

Amit, Maayan; Sela, Noa; Keren, Hadas; Melamed, Ze'ev; Muler, Inna; Shomron, Noam; Izraeli, Shai; Ast, Gil

2007-11-29

Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains.

Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene

PubMed Central

Amit, Maayan; Sela, Noa; Keren, Hadas; Melamed, Ze'ev; Muler, Inna; Shomron, Noam; Izraeli, Shai; Ast, Gil

2007-01-01

Background Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. Results Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. Conclusion The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains. PMID:18047649

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

PubMed Central

Kramer, Marianne C.; Liang, Dongming; Tatomer, Deirdre C.; Gold, Beth; March, Zachary M.; Cherry, Sara; Wilusz, Jeremy E.

2015-01-01

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3′ end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine–arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes. PMID:26450910

Exon Shuffling and Origin of Scorpion Venom Biodiversity

PubMed Central

Wang, Xueli; Gao, Bin; Zhu, Shunyi

2016-01-01

Scorpion venom is a complex combinatorial library of peptides and proteins with multiple biological functions. A combination of transcriptomic and proteomic techniques has revealed its enormous molecular diversity, as identified by the presence of a large number of ion channel-targeted neurotoxins with different folds, membrane-active antimicrobial peptides, proteases, and protease inhibitors. Although the biodiversity of scorpion venom has long been known, how it arises remains unsolved. In this work, we analyzed the exon-intron structures of an array of scorpion venom protein-encoding genes and unexpectedly found that nearly all of these genes possess a phase-1 intron (one intron located between the first and second nucleotides of a codon) near the cleavage site of a signal sequence despite their mature peptides remarkably differ. This observation matches a theory of exon shuffling in the origin of new genes and suggests that recruitment of different folds into scorpion venom might be achieved via shuffling between body protein-coding genes and ancestral venom gland-specific genes that presumably contributed tissue-specific regulatory elements and secretory signal sequences. PMID:28035955

Exon Shuffling and Origin of Scorpion Venom Biodiversity.

PubMed

Wang, Xueli; Gao, Bin; Zhu, Shunyi

2016-12-26

Scorpion venom is a complex combinatorial library of peptides and proteins with multiple biological functions. A combination of transcriptomic and proteomic techniques has revealed its enormous molecular diversity, as identified by the presence of a large number of ion channel-targeted neurotoxins with different folds, membrane-active antimicrobial peptides, proteases, and protease inhibitors. Although the biodiversity of scorpion venom has long been known, how it arises remains unsolved. In this work, we analyzed the exon-intron structures of an array of scorpion venom protein-encoding genes and unexpectedly found that nearly all of these genes possess a phase-1 intron (one intron located between the first and second nucleotides of a codon) near the cleavage site of a signal sequence despite their mature peptides remarkably differ. This observation matches a theory of exon shuffling in the origin of new genes and suggests that recruitment of different folds into scorpion venom might be achieved via shuffling between body protein-coding genes and ancestral venom gland-specific genes that presumably contributed tissue-specific regulatory elements and secretory signal sequences.

Smart Phones: Platform Enabling Modular, Chemical, Biological, and Explosives Sensing

DTIC Science & Technology

2013-07-01

Smart phones: Platform Enabling Modular, Chemical, Biological, and Explosives Sensing by Amethist S. Finch , Matthew Coppock, Justin R...Chemical, Biological, and Explosives Sensing Amethist S. Finch , Matthew Coppock, Justin R. Bickford, Marvin A. Conn, Thomas J. Proctor, and...Explosives Sensing 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Amethist S. Finch , Matthew Coppock, Justin R

A Modular Habitation System for Human Planetary and Space Exploration

NASA Technical Reports Server (NTRS)

Howe, A. Scott

2015-01-01

A small-diameter modular pressure vessel system is devised that can be applied to planetary surface and deep space human exploration missions. As one of the recommendations prepared for the NASA Human Spaceflight Architecture Team (HAT) Evolvable Mars Campaign (EMC), a compact modular system can provide a Mars-forward approach to a variety of missions and environments. Small cabins derived from the system can fit into the Space Launch System (SLS) Orion "trunk", or can be mounted with mobility systems to function as pressurized rovers, in-space taxis, ascent stage cabins, or propellant tanks. Larger volumes can be created using inflatable elements for long-duration deep space missions and planetary surface outposts. This paper discusses how a small-diameter modular system can address functional requirements, mass and volume constraints, and operational scenarios.

Mechanisms and Regulation of Alternative Pre-mRNA Splicing

PubMed Central

Lee, Yeon

2015-01-01

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16.

PubMed

Hamilton, Natasha A; Tammen, Imke; Raadsma, Herman W

2013-01-01

Angiotensin converting enzyme (ACE) is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D) polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs) discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

Multi-Species Comparative Analysis of the Equine ACE Gene Identifies a Highly Conserved Potential Transcription Factor Binding Site in Intron 16

PubMed Central

Hamilton, Natasha A.; Tammen, Imke; Raadsma, Herman W.

2013-01-01

Angiotensin converting enzyme (ACE) is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D) polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs) discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism. PMID:23408978

Model of brain activation predicts the neural collective influence map of the brain

PubMed Central

Morone, Flaviano; Roth, Kevin; Min, Byungjoon; Makse, Hernán A.

2017-01-01

Efficient complex systems have a modular structure, but modularity does not guarantee robustness, because efficiency also requires an ingenious interplay of the interacting modular components. The human brain is the elemental paradigm of an efficient robust modular system interconnected as a network of networks (NoN). Understanding the emergence of robustness in such modular architectures from the interconnections of its parts is a longstanding challenge that has concerned many scientists. Current models of dependencies in NoN inspired by the power grid express interactions among modules with fragile couplings that amplify even small shocks, thus preventing functionality. Therefore, we introduce a model of NoN to shape the pattern of brain activations to form a modular environment that is robust. The model predicts the map of neural collective influencers (NCIs) in the brain, through the optimization of the influence of the minimal set of essential nodes responsible for broadcasting information to the whole-brain NoN. Our results suggest intervention protocols to control brain activity by targeting influential neural nodes predicted by network theory. PMID:28351973

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

Kerr, J.M.; Fisher, L.W.; Termine, J.D.

The authors have isolated and partially sequenced the human bone sialoprotein gene (IBSP). IBSP has been sublocalized by in situ hybridization to chromosome 4q38-q31 and is composed of six small exons (51 to 159 bp) and 1 large exon ([approximately]2.6 kb). The intron/exon junctions defined by sequence analysis are of class O, retaining an intact coding triplet. Sequence analysis of the 5[prime] upstream region revealed a TATAA (nucleotides -30 to-25 from the transcriptional start point) and a CCAAT (nucleotides -56 to-52) box, both in the reverse orientation. Intron 1 contains interesting structural elements composed of polypyrimidine repeats followed by amore » poly(AC)[sub n] tract. Both types of structural elements have been detected in promoter regions of other genes and have been implicated in transcriptional regulation. Several differences between the previously published cDNA sequence and the authors' sequence have been identified, most of which are contained within the untranslated exon 1. Three base revisions in the coding region include a G to T (Gly to Val, amino acid 195), T to C (Val to Ala, amino acid 268), and T to A (Glu to Asp, amino acid 270). In conclusion, the genomic organization and potential regulatory elements of human IBSP have been elucidated. 42 refs., 4 figs., 1 tab.« less

Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter.

PubMed

Bao, X; Shorrosh, B S; Ohlrogge, J B

1997-11-01

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: Lesson learned from Anemonia viridis.

PubMed

Chi, Sylvia Ighem; Urbarova, Ilona; Johansen, Steinar D

2018-04-30

The mitochondrial genomes of sea anemones are dynamic in structure. Invasion by genetic elements, such as self-catalytic group I introns or insertion-like sequences, contribute to sea anemone mitochondrial genome expansion and complexity. By using next generation sequencing we investigated the complete mtDNAs and corresponding transcriptomes of the temperate sea anemone Anemonia viridis and its closer tropical relative Anemonia majano. Two versions of fused homing endonuclease gene (HEG) organization were observed among the Actiniidae sea anemones; in-frame gene fusion and pseudo-gene fusion. We provided support for the pseudo-gene fusion organization in Anemonia species, resulting in a repressed HEG from the COI-884 group I intron. orfA, a putative protein-coding gene with insertion-like features, was present in both Anemonia species. Interestingly, orfA and COI expression were significantly up-regulated upon long-term environmental stress corresponding to low seawater pH conditions. This study provides new insights to the dynamics of sea anemone mitochondrial genome structure and function. Copyright © 2018 Elsevier B.V. All rights reserved.

Cloning of a CACTA transposon-like insertion in intron I of tomato invertase Lin5 gene and identification of transposase-like sequences of Solanaceae species.

PubMed

Proels, Reinhard K; Roitsch, Thomas

2006-03-01

Very few CACTA transposon-like sequences have been described in Solanaceae species. Sequence information has been restricted to partial transposase (TPase)-like fragments, and no target gene of CACTA-like transposon insertion has been described in tomato to date. In this manuscript, we report on a CACTA transposon-like insertion in intron I of tomato (Lycopersicon esculentum) invertase gene Lin5 and TPase-like sequences of several Solanaceae species. Consensus primers deduced from the TPase region of the tomato CACTA transposon-like element allowed the amplification of similar sequences from various Solanaceae species of different subfamilies including Solaneae (Solanum tuberosum), Cestreae (Nicotiana tabacum) and Datureae (Datura stramonium). This demonstrates the ubiquitous presence of CACTA-like elements in Solanaceae genomes. The obtained partial sequences are highly conserved, and allow further detection and detailed analysis of CACTA-like transposons throughout Solanaceae species. CACTA-like transposon sequences make possible the evaluation of their use for genome analysis, functional studies of genes and the evolutionary relationships between plant species.

Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers.

PubMed

Krizek, Beth A

2015-10-12

The AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3) gene of Arabidopsis thaliana is a key regulator of growth and patterning in both shoots and roots. AIL6 encodes an AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor that is expressed in the root stem cell niche, the peripheral region of the shoot apical meristem and young lateral organ primordia. In flowers, AIL6 acts redundantly with AINTEGUMENTA (ANT) to regulate floral organ positioning, growth, identity and patterning. Experiments were undertaken to define the genomic regions required for AIL6 function and expression in flowers. Transgenic plants expressing a copy of the coding region of AIL6 in the context of 7.7 kb of 5' sequence and 919 bp of 3' sequence (AIL6:cAIL6-3') fail to fully complement AIL6 function when assayed in the ant-4 ail6-2 double mutant background. In contrast, a genomic copy of AIL6 with the same amount of 5' and 3' sequence (AIL6:gAIL6-3') can fully complement ant-4 ail6-2. In addition, a genomic copy of AIL6 with 590 bp of 5' sequence and 919 bp of 3' sequence (AIL6m:gAIL6-3') complements ant-4 ail6-2 and contains all regulatory elements needed to confer normal AIL6 expression in inflorescences. Efforts to map cis-regulatory elements reveal that the third intron of AIL6 contains enhancer elements that confer expression in young flowers but in a broader pattern than that of AIL6 mRNA in wild-type flowers. Some AIL6:gAIL6-3' and AIL6m:gAIL6-3' lines confer an over-rescue phenotype in the ant-4 ail6-2 background that is correlated with higher levels of AIL6 mRNA accumulation. The results presented here indicate that AIL6 intronic sequences serve as transcriptional enhancer elements. In addition, the results show that increased expression of AIL6 can partially compensate for loss of ANT function in flowers.

Elevated Rate of Fixation of Endogenous Retroviral Elements in Haplorhini TRIM5 and TRIM22 Genomic Sequences: Impact on Transcriptional Regulation

PubMed Central

Diehl, William E.; Johnson, Welkin E.; Hunter, Eric

2013-01-01

All genes in the TRIM6/TRIM34/TRIM5/TRIM22 locus are type I interferon inducible, with TRIM5 and TRIM22 possessing antiviral properties. Evolutionary studies involving the TRIM6/34/5/22 locus have predominantly focused on the coding sequence of the genes, finding that TRIM5 and TRIM22 have undergone high rates of both non-synonymous nucleotide replacements and in-frame insertions and deletions. We sought to understand if divergent evolutionary pressures on TRIM6/34/5/22 coding regions have selected for modifications in the non-coding regions of these genes and explore whether such non-coding changes may influence the biological function of these genes. The transcribed genomic regions, including the introns, of TRIM6, TRIM34, TRIM5, and TRIM22 from ten Haplorhini primates and one prosimian species were analyzed for transposable element content. In Haplorhini species, TRIM5 displayed an exaggerated interspecies variability, predominantly resulting from changes in the composition of transposable elements in the large first and fourth introns. Multiple lineage-specific endogenous retroviral long terminal repeats (LTRs) were identified in the first intron of TRIM5 and TRIM22. In the prosimian genome, we identified a duplication of TRIM5 with a concomitant loss of TRIM22. The transposable element content of the prosimian TRIM5 genes appears to largely represent the shared Haplorhini/prosimian ancestral state for this gene. Furthermore, we demonstrated that one such differentially fixed LTR provides for species-specific transcriptional regulation of TRIM22 in response to p53 activation. Our results identify a previously unrecognized source of species-specific variation in the antiviral TRIM genes, which can lead to alterations in their transcriptional regulation. These observations suggest that there has existed long-term pressure for exaptation of retroviral LTRs in the non-coding regions of these genes. This likely resulted from serial viral challenges and provided a mechanism for rapid alteration of transcriptional regulation. To our knowledge, this represents the first report of persistent evolutionary pressure for the capture of retroviral LTR insertions. PMID:23516500

Physiological Perturbation Reveals Modularity of Eyespot Development in the Painted Lady Butterfly, Vanessa cardui

PubMed Central

Rhen, Turk; Simmons, Rebecca B.

2016-01-01

Butterfly eyespots are complex morphological traits that can vary in size, shape and color composition even on the same wing surface. Homology among eyespots suggests they share a common developmental basis and function as an integrated unit in response to selection. Despite strong evidence of genetic integration, eyespots can also exhibit modularity or plasticity, indicating an underlying flexibility in pattern development. The extent to which particular eyespots or eyespot color elements exhibit modularity or integration is poorly understood, particularly following exposure to novel conditions. We used perturbation experiments to explore phenotypic correlations among different eyespots and their color elements on the ventral hindwing of V. cardui. Specifically, we identified which eyespots and eyespot features are most sensitive to perturbation by heat shock and injection of heparin—a cold shock mimic. For both treatments, the two central eyespots (3 + 4) were most affected by the experimental perturbations, whereas the outer eyespot border was more resistant to modification than the interior color elements. Overall, the individual color elements displayed a similar response to heat shock across all eyespots, but varied in their response to each other. Graphical modeling also revealed that although eyespots differ morphologically, regulation of eyespot size and colored elements appear to be largely integrated across the wing. Patterns of integration, however, were disrupted following heat shock, revealing that the strength of integration varies across the wing and is strongest between the two central eyespots. These findings support previous observations that document coupling between eyespots 3 + 4 in other nymphalid butterflies. PMID:27560365

Physiological Perturbation Reveals Modularity of Eyespot Development in the Painted Lady Butterfly, Vanessa cardui.

PubMed

Connahs, Heidi; Rhen, Turk; Simmons, Rebecca B

2016-01-01

Butterfly eyespots are complex morphological traits that can vary in size, shape and color composition even on the same wing surface. Homology among eyespots suggests they share a common developmental basis and function as an integrated unit in response to selection. Despite strong evidence of genetic integration, eyespots can also exhibit modularity or plasticity, indicating an underlying flexibility in pattern development. The extent to which particular eyespots or eyespot color elements exhibit modularity or integration is poorly understood, particularly following exposure to novel conditions. We used perturbation experiments to explore phenotypic correlations among different eyespots and their color elements on the ventral hindwing of V. cardui. Specifically, we identified which eyespots and eyespot features are most sensitive to perturbation by heat shock and injection of heparin-a cold shock mimic. For both treatments, the two central eyespots (3 + 4) were most affected by the experimental perturbations, whereas the outer eyespot border was more resistant to modification than the interior color elements. Overall, the individual color elements displayed a similar response to heat shock across all eyespots, but varied in their response to each other. Graphical modeling also revealed that although eyespots differ morphologically, regulation of eyespot size and colored elements appear to be largely integrated across the wing. Patterns of integration, however, were disrupted following heat shock, revealing that the strength of integration varies across the wing and is strongest between the two central eyespots. These findings support previous observations that document coupling between eyespots 3 + 4 in other nymphalid butterflies.

Regulation of the intronic promoter of rat estrogen receptor alpha gene, responsible for truncated estrogen receptor product-1 expression.

PubMed

Schausi, Diane; Tiffoche, Christophe; Thieulant, Marie-Lise

2003-07-01

We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

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

Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding tomore » the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.« less

Transcription Factor KLF5 Binds a Cyclin E1 Polymorphic Intronic Enhancer to Confer Increased Bladder Cancer Risk

PubMed Central

Pattison, Jillian M.; Posternak, Valeriya; Cole, Michael D.

2016-01-01

It is well established that environmental toxins, such as exposure to arsenic, are risk factors in the development of urinary bladder cancer, yet recent genome-wide association studies (GWAS) provide compelling evidence that there is a strong genetic component associated with disease predisposition. A single nucleotide polymorphism (SNP), rs8102137, was identified on chromosome 19q12, residing 6 kb upstream of the important cell cycle regulator and proto-oncogene, Cyclin E1 (CCNE1). However, the functional role of this variant in bladder cancer predisposition has been unclear since it lies within a non-coding region of the genome. Here, it is demonstrated that bladder cancer cells heterozygous for this SNP exhibit biased allelic expression of CCNE1 with 1.5-fold more transcription occurring from the risk allele. Furthermore, using chromatin immunoprecipitation assays, a novel enhancer element was identified within the first intron of CCNE1 that binds Kruppel-like Factor 5 (KLF5), a known transcriptional activator in bladder cancer. Moreover, the data reveal that the presence of rs200996365, a SNP in high linkage disequilibrium with rs8102137 residing in the center of a KLF5 motif, alters KLF5 binding to this genomic region. Through luciferase assays and CRISPR-Cas9 genome editing, a novel polymorphic intronic regulatory element controlling CCNE1 transcription is characterized. These studies uncover how a cancer-associated polymorphism mechanistically contributes to an increased predisposition for bladder cancer development. Implications A polymorphic KLF5 binding site near the CCNE1 gene explains genetic risk identified through genome wide association studies. PMID:27514407

Sculpting the Intrinsic Modular Organization of Spontaneous Brain Activity by Art.

PubMed

Lin, Chia-Shu; Liu, Yong; Huang, Wei-Yuan; Lu, Chia-Feng; Teng, Shin; Ju, Tzong-Ching; He, Yong; Wu, Yu-Te; Jiang, Tianzi; Hsieh, Jen-Chuen

2013-01-01

Artistic training is a complex learning that requires the meticulous orchestration of sophisticated polysensory, motor, cognitive, and emotional elements of mental capacity to harvest an aesthetic creation. In this study, we investigated the architecture of the resting-state functional connectivity networks from professional painters, dancers and pianists. Using a graph-based network analysis, we focused on the art-related changes of modular organization and functional hubs in the resting-state functional connectivity network. We report that the brain architecture of artists consists of a hierarchical modular organization where art-unique and artistic form-specific brain states collectively mirror the mind states of virtuosos. We show that even in the resting state, this type of extraordinary and long-lasting training can macroscopically imprint a neural network system of spontaneous activity in which the related brain regions become functionally and topologically modularized in both domain-general and domain-specific manners. The attuned modularity reflects a resilient plasticity nurtured by long-term experience.

Sculpting the Intrinsic Modular Organization of Spontaneous Brain Activity by Art

PubMed Central

Lin, Chia-Shu; Liu, Yong; Huang, Wei-Yuan; Lu, Chia-Feng; Teng, Shin; Ju, Tzong-Ching; He, Yong; Wu, Yu-Te; Jiang, Tianzi; Hsieh, Jen-Chuen

2013-01-01

Artistic training is a complex learning that requires the meticulous orchestration of sophisticated polysensory, motor, cognitive, and emotional elements of mental capacity to harvest an aesthetic creation. In this study, we investigated the architecture of the resting-state functional connectivity networks from professional painters, dancers and pianists. Using a graph-based network analysis, we focused on the art-related changes of modular organization and functional hubs in the resting-state functional connectivity network. We report that the brain architecture of artists consists of a hierarchical modular organization where art-unique and artistic form-specific brain states collectively mirror the mind states of virtuosos. We show that even in the resting state, this type of extraordinary and long-lasting training can macroscopically imprint a neural network system of spontaneous activity in which the related brain regions become functionally and topologically modularized in both domain-general and domain-specific manners. The attuned modularity reflects a resilient plasticity nurtured by long-term experience. PMID:23840527

Group I introns are widespread in archaea.

PubMed

Nawrocki, Eric P; Jones, Thomas A; Eddy, Sean R

2018-05-18

Group I catalytic introns have been found in bacterial, viral, organellar, and some eukaryotic genomes, but not in archaea. All known archaeal introns are bulge-helix-bulge (BHB) introns, with the exception of a few group II introns. It has been proposed that BHB introns arose from extinct group I intron ancestors, much like eukaryotic spliceosomal introns are thought to have descended from group II introns. However, group I introns have little sequence conservation, making them difficult to detect with standard sequence similarity searches. Taking advantage of recent improvements in a computational homology search method that accounts for both conserved sequence and RNA secondary structure, we have identified 39 group I introns in a wide range of archaeal phyla, including examples of group I introns and BHB introns in the same host gene.

Horizontal transfer and gene conversion as an important driving force in shaping the landscape of mitochondrial introns.

PubMed

Wu, Baojun; Hao, Weilong

2014-04-16

Group I introns are highly dynamic and mobile, featuring extensive presence-absence variation and widespread horizontal transfer. Group I introns can invade intron-lacking alleles via intron homing powered by their own encoded homing endonuclease gene (HEG) after horizontal transfer or via reverse splicing through an RNA intermediate. After successful invasion, the intron and HEG are subject to degeneration and sequential loss. It remains unclear whether these mechanisms can fully address the high dynamics and mobility of group I introns. Here, we found that HEGs undergo a fast gain-and-loss turnover comparable with introns in the yeast mitochondrial 21S-rRNA gene, which is unexpected, as the intron and HEG are generally believed to move together as a unit. We further observed extensively mosaic sequences in both the introns and HEGs, and evidence of gene conversion between HEG-containing and HEG-lacking introns. Our findings suggest horizontal transfer and gene conversion can accelerate HEG/intron degeneration and loss, or rescue and propagate HEG/introns, and ultimately result in high HEG/intron turnover rate. Given that up to 25% of the yeast mitochondrial genome is composed of introns and most mitochondrial introns are group I introns, horizontal transfer and gene conversion could have served as an important mechanism in introducing mitochondrial intron diversity, promoting intron mobility and consequently shaping mitochondrial genome architecture.

On the role of sparseness in the evolution of modularity in gene regulatory networks

PubMed Central

2018-01-01

Modularity is a widespread property in biological systems. It implies that interactions occur mainly within groups of system elements. A modular arrangement facilitates adjustment of one module without perturbing the rest of the system. Therefore, modularity of developmental mechanisms is a major factor for evolvability, the potential to produce beneficial variation from random genetic change. Understanding how modularity evolves in gene regulatory networks, that create the distinct gene activity patterns that characterize different parts of an organism, is key to developmental and evolutionary biology. One hypothesis for the evolution of modules suggests that interactions between some sets of genes become maladaptive when selection favours additional gene activity patterns. The removal of such interactions by selection would result in the formation of modules. A second hypothesis suggests that modularity evolves in response to sparseness, the scarcity of interactions within a system. Here I simulate the evolution of gene regulatory networks and analyse diverse experimentally sustained networks to study the relationship between sparseness and modularity. My results suggest that sparseness alone is neither sufficient nor necessary to explain modularity in gene regulatory networks. However, sparseness amplifies the effects of forms of selection that, like selection for additional gene activity patterns, already produce an increase in modularity. That evolution of new gene activity patterns is frequent across evolution also supports that it is a major factor in the evolution of modularity. That sparseness is widespread across gene regulatory networks indicates that it may have facilitated the evolution of modules in a wide variety of cases. PMID:29775459

On the role of sparseness in the evolution of modularity in gene regulatory networks.

PubMed

Espinosa-Soto, Carlos

2018-05-01

Modularity is a widespread property in biological systems. It implies that interactions occur mainly within groups of system elements. A modular arrangement facilitates adjustment of one module without perturbing the rest of the system. Therefore, modularity of developmental mechanisms is a major factor for evolvability, the potential to produce beneficial variation from random genetic change. Understanding how modularity evolves in gene regulatory networks, that create the distinct gene activity patterns that characterize different parts of an organism, is key to developmental and evolutionary biology. One hypothesis for the evolution of modules suggests that interactions between some sets of genes become maladaptive when selection favours additional gene activity patterns. The removal of such interactions by selection would result in the formation of modules. A second hypothesis suggests that modularity evolves in response to sparseness, the scarcity of interactions within a system. Here I simulate the evolution of gene regulatory networks and analyse diverse experimentally sustained networks to study the relationship between sparseness and modularity. My results suggest that sparseness alone is neither sufficient nor necessary to explain modularity in gene regulatory networks. However, sparseness amplifies the effects of forms of selection that, like selection for additional gene activity patterns, already produce an increase in modularity. That evolution of new gene activity patterns is frequent across evolution also supports that it is a major factor in the evolution of modularity. That sparseness is widespread across gene regulatory networks indicates that it may have facilitated the evolution of modules in a wide variety of cases.

Identification of human short introns

PubMed Central

Abebrese, Emmanuel L.; Arnold, Zachary R.; Armstrong, Katharine; Burns, Lindsay; Day, R. Thomas; Hsu, Daniel G.; Jarrell, Katherine; Luo, Yi; Mugayo, Daphine

2017-01-01

Canonical pre-mRNA splicing requires snRNPs and associated splicing factors to excise conserved intronic sequences, with a minimum intron length required for efficient splicing. Non-canonical splicing–intron excision without the spliceosome–has been documented; most notably, some tRNAs and the XBP1 mRNA contain short introns that are not removed by the spliceosome. There have been some efforts to identify additional short introns, but little is known about how many short introns are processed from mRNAs. Here, we report an approach to identify RNA short introns from RNA-Seq data, discriminating against small genomic deletions. We identify hundreds of short introns conserved among multiple human cell lines. These short introns are often alternatively spliced and are found in a variety of RNAs–both mRNAs and lncRNAs. Short intron splicing efficiency is increased by secondary structure, and we detect both canonical and non-canonical short introns. In many cases, splicing of these short introns from mRNAs is predicted to alter the reading frame and change protein output. Our findings imply that standard gene prediction models which often assume a lower limit for intron size fail to predict short introns effectively. We conclude that short introns are abundant in the human transcriptome, and short intron splicing represents an added layer to mRNA regulation. PMID:28520720

Forward osmosis membrane modular configurations for osmotic dilution of seawater by forward osmosis and reverse osmosis hybrid system.

PubMed

Kim, Jung Eun; Phuntsho, Sherub; Ali, Syed Muztuza; Choi, Joon Young; Shon, Ho Kyong

2018-01-01

This study evaluates various options for full-scale modular configuration of forward osmosis (FO) process for osmotic dilution of seawater using wastewater for simultaneous desalination and water reuse through FO-reverse osmosis (RO) hybrid system. Empirical relationship obtained from one FO membrane element operation was used to simulate the operational performances of different FO module configurations. The main limiting criteria for module operation is to always maintain the feed pressure higher than the draw pressure throughout the housing module for safe operation without affecting membrane integrity. Experimental studies under the conditions tested in this study show that a single membrane housing cannot accommodate more than four elements as the draw pressure exceeds the feed pressure. This then indicates that a single stage housing with eight elements is not likely to be practical for safe FO operation. Hence, six different FO modular configurations were proposed and simulated. A two-stage FO configuration with multiple housings (in parallel) in the second stage using same or larger spacer thickness reduces draw pressure build-up as the draw flow rates are reduced to half in the second stage thereby allowing more than four elements in the second stage housing. The loss of feed pressure (pressure drop) and osmotic driving force in the second stage are compensated by operating under the pressure assisted osmosis (PAO) mode, which helps enhance permeate flux and maintains positive pressure differences between the feed and draw chamber. The PAO energy penalty is compensated by enhanced permeate throughput, reduced membrane area, and plant footprint. The contribution of FO/PAO to total energy consumption was not significant compared to post RO desalination (90%) indicating that the proposed two-stage FO modular configuration is one way of making the FO full-scale operation practical for FO-RO hybrid system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Occurrence of Can-SINEs and intron sequence evolution supports robust phylogeny of pinniped carnivores and their terrestrial relatives.

PubMed

Schröder, Christiane; Bleidorn, Christoph; Hartmann, Stefanie; Tiedemann, Ralph

2009-12-15

Investigating the dog genome we found 178965 introns with a moderate length of 200-1000 bp. A screening of these sequences against 23 different repeat libraries to find insertions of short interspersed elements (SINEs) detected 45276 SINEs. Virtually all of these SINEs (98%) belong to the tRNA-derived Can-SINE family. Can-SINEs arose about 55 million years ago before Carnivora split into two basal groups, the Caniformia (dog-like carnivores) and the Feliformia (cat-like carnivores). Genome comparisons of dog and cat recovered 506 putatively informative SINE loci for caniformian phylogeny. In this study we show how to use such genome information of model organisms to research the phylogeny of related non-model species of interest. Investigating a dataset including representatives of all major caniformian lineages, we analysed 24 randomly chosen loci for 22 taxa. All loci were amplifiable and revealed 17 parsimony-informative SINE insertions. The screening for informative SINE insertions yields a large amount of sequence information, in particular of introns, which contain reliable phylogenetic information as well. A phylogenetic analysis of intron- and SINE sequence data provided a statistically robust phylogeny which is congruent with the absence/presence pattern of our SINE markers. This phylogeny strongly supports a sistergroup relationship of Musteloidea and Pinnipedia. Within Pinnipedia, we see strong support from bootstrapping and the presence of a SINE insertion for a sistergroup relationship of the walrus with the Otariidae.

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins.

PubMed

Kramer, Marianne C; Liang, Dongming; Tatomer, Deirdre C; Gold, Beth; March, Zachary M; Cherry, Sara; Wilusz, Jeremy E

2015-10-15

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼ 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes. © 2015 Kramer et al.; Published by Cold Spring Harbor Laboratory Press.

ENVIRONMENTAL RESEARCH BRIEF : ANALYTIC ELEMENT MODELING OF GROUND-WATER FLOW AND HIGH PERFORMANCE COMPUTING

EPA Science Inventory

Several advances in the analytic element method have been made to enhance its performance and facilitate three-dimensional ground-water flow modeling in a regional aquifer setting. First, a new public domain modular code (ModAEM) has been developed for modeling ground-water flow ...

Splicing-Related Features of Introns Serve to Propel Evolution

PubMed Central

Luo, Yuping; Li, Chun; Gong, Xi; Wang, Yanlu; Zhang, Kunshan; Cui, Yaru; Sun, Yi Eve; Li, Siguang

2013-01-01

The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron's ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution. PMID:23516505

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

PubMed Central

Lakshmi, G. Girija; Ghosh, Sushmita; Jones, Gabriel P.; Parikh, Roshni; Rawlins, Bridgette A.; Vaughn, Jack C.

2014-01-01

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4,000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA-protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model. PMID:23026215

Introns: The Functional Benefits of Introns in Genomes.

PubMed

Jo, Bong-Seok; Choi, Sun Shim

2015-12-01

The intron has been a big biological mystery since it was first discovered in several aspects. First, all of the completely sequenced eukaryotes harbor introns in the genomic structure, whereas no prokaryotes identified so far carry introns. Second, the amount of total introns varies in different species. Third, the length and number of introns vary in different genes, even within the same species genome. Fourth, all introns are copied into RNAs by transcription and DNAs by replication processes, but intron sequences do not participate in protein-coding sequences. The existence of introns in the genome should be a burden to some cells, because cells have to consume a great deal of energy to copy and excise them exactly at the correct positions with the help of complicated spliceosomal machineries. The existence throughout the long evolutionary history is explained, only if selective advantages of carrying introns are assumed to be given to cells to overcome the negative effect of introns. In that regard, we summarize previous research about the functional roles or benefits of introns. Additionally, several other studies strongly suggesting that introns should not be junk will be introduced.

Antisense Oligonucleotides Modulating Activation of a Nonsense-Mediated RNA Decay Switch Exon in the ATM Gene.

PubMed

Kralovicova, Jana; Moreno, Pedro M D; Cross, Nicholas C P; Pêgo, Ana Paula; Vorechovsky, Igor

2016-12-01

ATM (ataxia-telangiectasia, mutated) is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs.

Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

NASA Astrophysics Data System (ADS)

Li, Shengjie; Bai, Junjie; Wang, Lin

2008-08-01

Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

Identification of 15 novel partial SHOX deletions and 13 partial duplications, and a review of the literature reveals intron 3 to be a hotspot region.

PubMed

Benito-Sanz, Sara; Belinchon-Martínez, Alberta; Aza-Carmona, Miriam; de la Torre, Carolina; Huber, Celine; González-Casado, Isabel; Ross, Judith L; Thomas, N Simon; Zinn, Andrew R; Cormier-Daire, Valerie; Heath, Karen E

2017-02-01

Short stature homeobox gene (SHOX) is located in the pseudoautosomal region 1 of the sex chromosomes. It encodes a transcription factor implicated in the skeletal growth. Point mutations, deletions or duplications of SHOX or its transcriptional regulatory elements are associated with two skeletal dysplasias, Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), as well as in a small proportion of idiopathic short stature (ISS) individuals. We have identified a total of 15 partial SHOX deletions and 13 partial SHOX duplications in LWD, LMD and ISS patients referred for routine SHOX diagnostics during a 10 year period (2004-2014). Subsequently, we characterized these alterations using MLPA (multiplex ligation-dependent probe amplification assay), fine-tiling array CGH (comparative genomic hybridation) and breakpoint PCR. Nearly half of the alterations have a distal or proximal breakpoint in intron 3. Evaluation of our data and that in the literature reveals that although partial deletions and duplications only account for a small fraction of SHOX alterations, intron 3 appears to be a breakpoint hotspot, with alterations arising by non-allelic homologous recombination, non-homologous end joining or other complex mechanisms.

Introduction to COFFE: The Next-Generation HPCMP CREATE-AV CFD Solver

NASA Technical Reports Server (NTRS)

Glasby, Ryan S.; Erwin, J. Taylor; Stefanski, Douglas L.; Allmaras, Steven R.; Galbraith, Marshall C.; Anderson, W. Kyle; Nichols, Robert H.

2016-01-01

HPCMP CREATE-AV Conservative Field Finite Element (COFFE) is a modular, extensible, robust numerical solver for the Navier-Stokes equations that invokes modularity and extensibility from its first principles. COFFE implores a flexible, class-based hierarchy that provides a modular approach consisting of discretization, physics, parallelization, and linear algebra components. These components are developed with modern software engineering principles to ensure ease of uptake from a user's or developer's perspective. The Streamwise Upwind/Petrov-Galerkin (SU/PG) method is utilized to discretize the compressible Reynolds-Averaged Navier-Stokes (RANS) equations tightly coupled with a variety of turbulence models. The mathematics and the philosophy of the methodology that makes up COFFE are presented.

Digital Material Assembly by Passive Means and Modular Isotropic Lattice Extruder System

NASA Technical Reports Server (NTRS)

Gershenfeld, Neil (Inventor); Carney, Matthew Eli (Inventor); Jenett, Benjamin (Inventor)

2017-01-01

A set of machines and related systems build structures by the additive assembly of discrete parts. These digital material assemblies constrain the constituent parts to a discrete set of possible positions and orientations. In doing so, the structures exhibit many of the properties inherent in digital communication such as error correction, fault tolerance and allow the assembly of precise structures with comparatively imprecise tools. Assembly of discrete cellular lattices by a Modular Isotropic Lattice Extruder System (MILES) is implemented by pulling strings of lattice elements through a forming die that enforces geometry constraints that lock the elements into a rigid structure that can then be pushed against and extruded out of the die as an assembled, loadbearing structure.

Recurrent Loss of Specific Introns during Angiosperm Evolution

PubMed Central

Wang, Hao; Devos, Katrien M.; Bennetzen, Jeffrey L.

2014-01-01

Numerous instances of presence/absence variations for introns have been documented in eukaryotes, and some cases of recurrent loss of the same intron have been suggested. However, there has been no comprehensive or phylogenetically deep analysis of recurrent intron loss. Of 883 cases of intron presence/absence variation that we detected in five sequenced grass genomes, 93 were confirmed as recurrent losses and the rest could be explained by single losses (652) or single gains (118). No case of recurrent intron gain was observed. Deep phylogenetic analysis often indicated that apparent intron gains were actually numerous independent losses of the same intron. Recurrent loss exhibited extreme non-randomness, in that some introns were removed independently in many lineages. The two larger genomes, maize and sorghum, were found to have a higher rate of both recurrent loss and overall loss and/or gain than foxtail millet, rice or Brachypodium. Adjacent introns and small introns were found to be preferentially lost. Intron loss genes exhibited a high frequency of germ line or early embryogenesis expression. In addition, flanking exon A+T-richness and intron TG/CG ratios were higher in retained introns. This last result suggests that epigenetic status, as evidenced by a loss of methylated CG dinucleotides, may play a role in the process of intron loss. This study provides the first comprehensive analysis of recurrent intron loss, makes a series of novel findings on the patterns of recurrent intron loss during the evolution of the grass family, and provides insight into the molecular mechanism(s) underlying intron loss. PMID:25474210

An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

PubMed Central

Ryu, Byoung Y.; Evans-Galea, Marguerite V.; Gray, John T.; Bodine, David M.; Persons, Derek A.

2008-01-01

Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a γ-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A γ-retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation. PMID:17991809

Force Structure: Preliminary Observations on Army Plans to Implement and Fund Modular Forces

DTIC Science & Technology

2005-03-16

affect the size and composition of the modular force as well as its cost. First, the Army’s Campaign Plan calls for a potential decision by fiscal year...upgrading existing active brigades, or other support and command elements; and • accounted for construction of temporary , relocatable facilities...Kevin Handley, Joah Iannotta, Harry Jobes, Joseph Kirschbaum, Eric Theus, Jason Venner , and J. Andrew Walker.Page 11 GAO-05-443T (350683) GAO’s

Advanced Modular Power Approach to Affordable, Supportable Space Systems

NASA Technical Reports Server (NTRS)

Oeftering, Richard C.; Kimnach, Greg L.; Fincannon, James; Mckissock,, Barbara I.; Loyselle, Patricia L.; Wong, Edmond

2013-01-01

Recent studies of missions to the Moon, Mars and Near Earth Asteroids (NEA) indicate that these missions often involve several distinct separately launched vehicles that must ultimately be integrated together in-flight and operate as one unit. Therefore, it is important to see these vehicles as elements of a larger segmented spacecraft rather than separate spacecraft flying in formation. The evolution of large multi-vehicle exploration architecture creates the need (and opportunity) to establish a global power architecture that is common across all vehicles. The Advanced Exploration Systems (AES) Modular Power System (AMPS) project managed by NASA Glenn Research Center (GRC) is aimed at establishing the modular power system architecture that will enable power systems to be built from a common set of modular building blocks. The project is developing, demonstrating and evaluating key modular power technologies that are expected to minimize non-recurring development costs, reduce recurring integration costs, as well as, mission operational and support costs. Further, modular power is expected to enhance mission flexibility, vehicle reliability, scalability and overall mission supportability. The AMPS project not only supports multi-vehicle architectures but should enable multi-mission capability as well. The AMPS technology development involves near term demonstrations involving developmental prototype vehicles and field demonstrations. These operational demonstrations not only serve as a means of evaluating modular technology but also provide feedback to developers that assure that they progress toward truly flexible and operationally supportable modular power architecture.

Origin and evolution of spliceosomal introns

PubMed Central

2012-01-01

Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded ‘introns first’ held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers’ Reports section. PMID:22507701

Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

PubMed Central

Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

2015-01-01

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

Evaluation of the mechanisms of intron loss and gain in the social amoebae Dictyostelium.

PubMed

Ma, Ming-Yue; Che, Xun-Ru; Porceddu, Andrea; Niu, Deng-Ke

2015-12-18

Spliceosomal introns are a common feature of eukaryotic genomes. To approach a comprehensive understanding of intron evolution on Earth, studies should look beyond repeatedly studied groups such as animals, plants, and fungi. The slime mold Dictyostelium belongs to a supergroup of eukaryotes not covered in previous studies. We found 441 precise intron losses in Dictyostelium discoideum and 202 precise intron losses in Dictyostelium purpureum. Consistent with these observations, Dictyostelium discoideum was found to have significantly more copies of reverse transcriptase genes than Dictyostelium purpureum. We also found that the lost introns are significantly further from the 5' end of genes than the conserved introns. Adjacent introns were prone to be lost simultaneously in Dictyostelium discoideum. In both Dictyostelium species, the exonic sequences flanking lost introns were found to have a significantly higher GC content than those flanking conserved introns. Together, these observations support a reverse-transcription model of intron loss in which intron losses were caused by gene conversion between genomic DNA and cDNA reverse transcribed from mature mRNA. We also identified two imprecise intron losses in Dictyostelium discoideum that may have resulted from genomic deletions. Ninety-eight putative intron gains were also observed. Consistent with previous studies of other lineages, the source sequences were found in only a small number of cases, with only two instances of intron gain identified in Dictyostelium discoideum. Although they diverged very early from animals and fungi, Dictyostelium species have similar mechanisms of intron loss.

The structure of the coding and 5'-flanking region of the type 1 iodothyronine deiodinase (dio1) gene is normal in a patient with suspected congenital dio1 deficiency.

PubMed

Toyoda, N; Kleinhaus, N; Larsen, P R

1996-06-01

We analyzed the exon-intron structure of the human type 1 deiodinase gene (dio1) and compared it with that of a patient with suspected congenital type 1 deiodinase (D1) deficiency. The hdio1 gene is identical in exon-intron arrangement to the mouse gene, with coding sequences and a selenocysteine insertion sequence (SECIS) element contained in four exons. There were no mutations in the sequences of exons 1-4 of the patient's genomic DNA. Functional studies by transient expression techniques showed no difference in basal promoter activity or T3 responsiveness between the patient's and the normal dio1 gene. A structural abnormality in the dio1 gene is not a likely explanation for this patient's D1-deficient phenotype.

Structure and composition of the courtship phenotype in the bird of paradise Parotia lawesii (Aves: Paradisaeidae).

PubMed

Scholes, Edwin

2008-01-01

Ethology is rooted in the idea that behavior is composed of discrete units and sub-units that can be compared among taxa in a phylogenetic framework. This means that behavior, like morphology and genes, is inherently modular. Yet, the concept of modularity is not well integrated into how we envision the behavioral components of phenotype. Understanding ethological modularity, and its implications for animal phenotype organization and evolution, requires that we construct interpretive schemes that permit us to examine it. In this study, I describe the structure and composition of a complex part of the behavioral phenotype of Parotia lawesii Ramsay, 1885--a bird of paradise (Aves: Paradisaeidae) from the forests of eastern New Guinea. I use archived voucher video clips, photographic ethograms, and phenotype ontology diagrams to describe the modular units comprising courtship at various levels of integration. Results show P. lawesii to have 15 courtship and mating behaviors (11 males, 4 females) hierarchically arranged within a complex seven-level structure. At the finest level examined, male displays are comprised of 49 modular sub-units (elements) differentially employed to form more complex modular units (phases and versions) at higher-levels of integration. With its emphasis on hierarchical modularity, this study provides an important conceptual framework for understanding courtship-related phenotypic complexity and provides a solid basis for comparative study of the genus Parotia.

Transcription regulation by distal enhancers

PubMed Central

Stadhouders, Ralph; van den Heuvel, Anita; Kolovos, Petros; Jorna, Ruud; Leslie, Kris; Grosveld, Frank; Soler, Eric

2012-01-01

Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system. PMID:22771987

Activation of farnesoid X receptor induces RECK expression in mouse liver

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

Peng, Xiaomin; Wu, Weibin; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032

2014-01-03

Highlights: •RECK is a novel transcriptional target gene of FXR in mouse liver. •The FXR response element is located within the intron 1 of RECK gene. •FXR agonist reverses the down-regulation of RECK in the liver in mouse NASH model. -- Abstract: Farnesoid X receptor (FXR) belongs to the ligand-activated nuclear receptor superfamily, and functions as a transcription factor regulating the transcription of numerous genes involved in bile acid homeostasis, lipoprotein and glucose metabolism. In the present study, we identified RECK, a membrane-anchored inhibitor of matrix metalloproteinases, as a novel target gene of FXR in mouse liver. We found thatmore » FXR agonist substantially augmented hepatic RECK mRNA and protein expression in vivo and in vitro. FXR regulated the transcription of RECK through directly binding to FXR response element located within intron 1 of the mouse RECK gene. Moreover, FXR agonist reversed the down-regulation of RECK in the livers from mice fed a methionine and choline deficient diet. In summary, our data suggest that RECK is a novel transcriptional target of FXR in mouse liver, and provide clues to better understanding the function of FXR in liver.« less

Gene organization and alternative splicing of human prohormone convertase PC8.

PubMed Central

Goodge, K A; Thomas, R J; Martin, T J; Gillespie, M T

1998-01-01

The mammalian Ca2+-dependent serine protease prohormone convertase PC8 is expressed ubiquitously, being transcribed as 3.5, 4.3 and 6.0 kb mRNA isoforms in various tissues. To determine the origin of these various mRNA isoforms we report the characterization of the human PC8 gene, which has been previously localized to chromosome 11q23-24. Consisting of 16 exons, the human PC8 gene spans approx. 27 kb. A comparison of the position of intron-exon junctions of the human PC8 gene with the gene structures of previously reported prohormone convertase genes demonstrated a divergence of the human PC8 from the highly conserved nature of the gene organization of this enzyme family. The nucleotide sequence of the 5'-flanking region of the human PC8 is reported and possesses putative promoter elements characteristic of a GC-rich promoter. Further supporting the potential role of a GC-rich promoter element, multiple transcriptional initiation sites within a 200 bp region were demonstrated. We propose that the various mRNA isoforms of PC8 result from the inclusion of intronic sequences within transcripts. PMID:9820811

Floating Double Deck Pier Fenders

DTIC Science & Technology

2011-07-01

Center FDDP Floating Double Deck Pier FEM Finite Element Model MHP Modular Hybrid Pier NAVFAC Naval Facilities RDT&E Research, Development, Testing...4. FEM Performance of MV1000x900B Elements ........................................................ 14 Figure 4-5. Biaxial UE1200x1200E3.1 Fender...Deflection .......................................................... 15 Figure 4-6. FEM Performance of Biaxial UE Fender

Analysis of nonuniformity in intron phase distribution.

PubMed Central

Fedorov, A; Suboch, G; Bujakov, M; Fedorova, L

1992-01-01

The distribution of different intron groups with respect to phases has been analyzed. It has been established that group II introns and nuclear introns have a minimum frequency of phase 2 introns. Since the phase of introns is an extremely conservative measure the observed minimum reflects evolutionary processes. A sample of all known, group I introns was too small to provide a valid characteristic of their phase distribution. The findings observed for the unequal distribution of phases cannot be explained solely on the basis of the mobile properties of introns. One of the most likely explanations for this nonuniformity in the intron phase distribution is the process of exon shuffling. It is proposed that group II introns originated at the early stages of evolution and were involved in the process of exon shuffling. PMID:1598214

Tissue- and case-specific retention of intron 40 in mature dystrophin mRNA.

PubMed

Nishida, Atsushi; Minegishi, Maki; Takeuchi, Atsuko; Niba, Emma Tabe Eko; Awano, Hiroyuki; Lee, Tomoko; Iijima, Kazumoto; Takeshima, Yasuhiro; Matsuo, Masafumi

2015-06-01

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined intron retention in dystrophin introns by reverse-transcription PCR from skeletal muscle, focusing on the nine shortest (all <1000 bp), because these are more likely to be retained. Only one, intron 40, was retained in mRNA; sequencing revealed insertion of a complete intron 40 (851 nt) between exons 40 and 41. The intron 40 retention product accounted for 1.2% of the total product but had a premature stop codon at the fifth intronic codon. Intron 40 retention was most strongly observed in the kidney (36.6%) and was not obtained from the fetal liver, lung, spleen or placenta. This indicated that intron retention is a tissue-specific event whose level varies among tissues. In two DMD patients, intron 40 retention was observed in one patient but not in the other. Examination of splicing regulatory factors revealed that intron 40 had the highest guanine-cytosine content of all examined introns in a 30-nt segment at its 3' end. Further studies are needed to clarify the biological role of intron 40-retained dystrophin mRNA.

Functional architecture of Escherichia coli: new insights provided by a natural decomposition approach.

PubMed

Freyre-González, Julio A; Alonso-Pavón, José A; Treviño-Quintanilla, Luis G; Collado-Vides, Julio

2008-10-27

Previous studies have used different methods in an effort to extract the modular organization of transcriptional regulatory networks. However, these approaches are not natural, as they try to cluster strongly connected genes into a module or locate known pleiotropic transcription factors in lower hierarchical layers. Here, we unravel the transcriptional regulatory network of Escherichia coli by separating it into its key elements, thus revealing its natural organization. We also present a mathematical criterion, based on the topological features of the transcriptional regulatory network, to classify the network elements into one of two possible classes: hierarchical or modular genes. We found that modular genes are clustered into physiologically correlated groups validated by a statistical analysis of the enrichment of the functional classes. Hierarchical genes encode transcription factors responsible for coordinating module responses based on general interest signals. Hierarchical elements correlate highly with the previously studied global regulators, suggesting that this could be the first mathematical method to identify global regulators. We identified a new element in transcriptional regulatory networks never described before: intermodular genes. These are structural genes that integrate, at the promoter level, signals coming from different modules, and therefore from different physiological responses. Using the concept of pleiotropy, we have reconstructed the hierarchy of the network and discuss the role of feedforward motifs in shaping the hierarchical backbone of the transcriptional regulatory network. This study sheds new light on the design principles underpinning the organization of transcriptional regulatory networks, showing a novel nonpyramidal architecture composed of independent modules globally governed by hierarchical transcription factors, whose responses are integrated by intermodular genes.

An intronic open reading frame was released from one of group II introns in the mitochondrial genome of the haptophyte Chrysochromulina sp. NIES-1333

PubMed Central

Nishimura, Yuki; Kamikawa, Ryoma; Hashimoto, Tetsuo; Inagaki, Yuji

2014-01-01

Mitochondrial (mt) genome sequences, which often bear introns, have been sampled from phylogenetically diverse eukaryotes. Thus, we can anticipate novel insights into intron evolution from previously unstudied mt genomes. We here investigated the origins and evolution of three introns in the mt genome of the haptophyte Chrysochromulina sp. NIES-1333, which was sequenced completely in this study. All the three introns were characterized as group II, on the basis of predicted secondary structure, and the conserved sequence motifs at the 5′ and 3′ termini. Our comparative studies on diverse mt genomes prompt us to propose that the Chrysochromulina mt genome laterally acquired the introns from mt genomes in distantly related eukaryotes. Many group II introns harbor intronic open reading frames for the proteins (intron-encoded proteins or IEPs), which likely facilitate the splicing of their host introns. However, we propose that a “free-standing,” IEP-like protein, which is not encoded within any introns in the Chrysochromulina mt genome, is involved in the splicing of the first cox1 intron that lacks any open reading frames. PMID:25054084

A modular finite-element model (MODFE) for areal and axisymmetric ground-water-flow problems, Part 3: Design philosophy and programming details

USGS Publications Warehouse

Torak, L.J.

1993-01-01

A MODular Finite-Element, digital-computer program (MODFE) was developed to simulate steady or unsteady-state, two-dimensional or axisymmetric ground-water-flow. The modular structure of MODFE places the computationally independent tasks that are performed routinely by digital-computer programs simulating ground-water flow into separate subroutines, which are executed from the main program by control statements. Each subroutine consists of complete sets of computations, or modules, which are identified by comment statements, and can be modified by the user without affecting unrelated computations elsewhere in the program. Simulation capabilities can be added or modified by either adding or modifying subroutines that perform specific computational tasks, and the modular-program structure allows the user to create versions of MODFE that contain only the simulation capabilities that pertain to the ground-water problem of interest. MODFE is written in a Fortran programming language that makes it virtually device independent and compatible with desk-top personal computers and large mainframes. MODFE uses computer storage and execution time efficiently by taking advantage of symmetry and sparseness within the coefficient matrices of the finite-element equations. Parts of the matrix coefficients are computed and stored as single-subscripted variables, which are assembled into a complete coefficient just prior to solution. Computer storage is reused during simulation to decrease storage requirements. Descriptions of subroutines that execute the computational steps of the modular-program structure are given in tables that cross reference the subroutines with particular versions of MODFE. Programming details of linear and nonlinear hydrologic terms are provided. Structure diagrams for the main programs show the order in which subroutines are executed for each version and illustrate some of the linear and nonlinear versions of MODFE that are possible. Computational aspects of changing stresses and boundary conditions with time and of mass-balance and error terms are given for each hydrologic feature. Program variables are listed and defined according to their occurrence in the main programs and in subroutines. Listings of the main programs and subroutines are given.

Three distinct modes of intron dynamics in the evolution of eukaryotes.

PubMed

Carmel, Liran; Wolf, Yuri I; Rogozin, Igor B; Koonin, Eugene V

2007-07-01

Several contrasting scenarios have been proposed for the origin and evolution of spliceosomal introns, a hallmark of eukaryotic genes. A comprehensive probabilistic model to obtain a definitive reconstruction of intron evolution was developed and applied to 391 sets of conserved genes from 19 eukaryotic species. It is inferred that a relatively high intron density was reached early, i.e., the last common ancestor of eukaryotes contained >2.15 introns/kilobase, and the last common ancestor of multicellular life forms harbored approximately 3.4 introns/kilobase, a greater intron density than in most of the extant fungi and in some animals. The rates of intron gain and intron loss appear to have been dropping during the last approximately 1.3 billion years, with the decline in the gain rate being much steeper. Eukaryotic lineages exhibit three distinct modes of evolution of the intron-exon structure. The primary, balanced mode, apparently, operates in all lineages. In this mode, intron gain and loss are strongly and positively correlated, in contrast to previous reports on inverse correlation between these processes. The second mode involves an elevated rate of intron loss and is prevalent in several lineages, such as fungi and insects. The third mode, characterized by elevated rate of intron gain, is seen only in deep branches of the tree, indicating that bursts of intron invasion occurred at key points in eukaryotic evolution, such as the origin of animals. Intron dynamics could depend on multiple mechanisms, and in the balanced mode, gain and loss of introns might share common mechanistic features.

Report on the Audit of the Acquisition of the Tactical Air Operations Center/Modular Control Equipment

DTIC Science & Technology

1991-06-06

This is our final report on the Audit of the Acquisition of the Tactical Air Operations Center/Modular Control Equipment (TAOC/MCE) for your...matters of concern that could affect the acquisition of the TAOC/MCE. We performed the audit from March through December 1990. The audit objective was...controls related to the audit objectives. The audit was made in accordance with the Inspector General’s critical program management element approach

New Approach to Road Construction in Oil-Producing Regions of Western Siberia

NASA Astrophysics Data System (ADS)

Piirainen, V. Y.; Estrin, Y.

2017-10-01

This article presents, as a polemic exercise, a new approach to road construction in marshland areas of oil and gas producing regions of Western Siberia. The approach is based on the use of novel modular elements that can be assembled into an integral structure by means of topological interlocking. The use of modern superlight concrete in conjunction with the new design systems based on the modular principle opens up new avenues to solving problems of road construction in regions with unstable, boggy soils.

Dynamic evolution of plant mitochondrial genomes: Mobile genes and introns and highly variable mutation rates

PubMed Central

Palmer, Jeffrey D.; Adams, Keith L.; Cho, Yangrae; Parkinson, Christopher L.; Qiu, Yin-Long; Song, Keming

2000-01-01

We summarize our recent studies showing that angiosperm mitochondrial (mt) genomes have experienced remarkably high rates of gene loss and concomitant transfer to the nucleus and of intron acquisition by horizontal transfer. Moreover, we find substantial lineage-specific variation in rates of these structural mutations and also point mutations. These findings mostly arise from a Southern blot survey of gene and intron distribution in 281 diverse angiosperms. These blots reveal numerous losses of mt ribosomal protein genes but, with one exception, only rare loss of respiratory genes. Some lineages of angiosperms have kept all of their mt ribosomal protein genes whereas others have lost most of them. These many losses appear to reflect remarkably high (and variable) rates of functional transfer of mt ribosomal protein genes to the nucleus in angiosperms. The recent transfer of cox2 to the nucleus in legumes provides both an example of interorganellar gene transfer in action and a starting point for discussion of the roles of mechanistic and selective forces in determining the distribution of genetic labor between organellar and nuclear genomes. Plant mt genomes also acquire sequences by horizontal transfer. A striking example of this is a homing group I intron in the mt cox1 gene. This extraordinarily invasive mobile element has probably been acquired over 1,000 times separately during angiosperm evolution via a recent wave of cross-species horizontal transfers. Finally, whereas all previously examined angiosperm mtDNAs have low rates of synonymous substitutions, mtDNAs of two distantly related angiosperms have highly accelerated substitution rates. PMID:10860957

An antisense RNA in a lytic cyanophage links psbA to a gene encoding a homing endonuclease.

PubMed

Millard, Andrew D; Gierga, Gregor; Clokie, Martha R J; Evans, David J; Hess, Wolfgang R; Scanlan, David J

2010-09-01

Cyanophage genomes frequently possess the psbA gene, encoding the D1 polypeptide of photosystem II. This protein is believed to maintain host photosynthetic capacity during infection and enhance phage fitness under high-light conditions. Although the first documented cyanophage-encoded psbA gene contained a group I intron, this feature has not been widely reported since, despite a plethora of new sequences becoming available. In this study, we show that in cyanophage S-PM2, this intron is spliced during the entire infection cycle. Furthermore, we report the widespread occurrence of psbA introns in marine metagenomic libraries, and with psbA often adjacent to a homing endonuclease (HE). Bioinformatic analysis of the intergenic region between psbA and the adjacent HE gene F-CphI in S-PM2 showed the presence of an antisense RNA (asRNA) connecting these two separate genetic elements. The asRNA is co-regulated with psbA and F-CphI, suggesting its involvement with their expression. Analysis of scaffolds from global ocean survey datasets shows this asRNA to be commonly associated with the 3' end of cyanophage psbA genes, implying that this potential mechanism of regulating marine 'viral' photosynthesis is evolutionarily conserved. Although antisense transcription is commonly found in eukaryotic and increasingly also in prokaryotic organisms, there has been no indication for asRNAs in lytic phages so far. We propose that this asRNA also provides a means of preventing the formation of mobile group I introns within cyanophage psbA genes.

Preliminary design study. Shuttle modular scanning spectroradiometer

NASA Technical Reports Server (NTRS)

1975-01-01

Fundamental concepts on which to base a detailed design for a Shuttle Modular Scanning Spectroradiometer were developed, and a preliminary design is presented. The recommended design features modularity and flexibility. It includes a 75-cm f/1.7-telescope assembly in an all-reflective Schmidt configuration, a solid state scan system (pushbroom) with high resolution over a 15 deg field of view, and ten detector channels covering the spectral range from 0.45 to 12.5 micrometers. It uses charge transfer device techniques to accommodate a large number of detector elements for earth observation measurements. Methods for in-flight radiometric calibration, for image motion compensation, and for data processing are described. Recommendations for ground support equipment are included, and interfaces with the shuttle orbiter vehicle are illustrated.

Modular thrust subsystem approaches to solar electric propulsion module design

NASA Technical Reports Server (NTRS)

Cake, J. E.; Sharp, G. R.; Oglebay, J. C.; Shaker, F. J.; Zavesky, R. J.

1976-01-01

Three approaches are presented for packaging the elements of a 30 cm ion thruster subsystem into a modular thrust subsystem. The individual modules, when integrated into a conceptual solar electric propulsion module are applicable to a multimission set of interplanetary flights with the space shuttle interim upper stage as the launch vehicle. The emphasis is on the structural and thermal integration of the components into the modular thrust subsystems. Thermal control for the power processing units is either by direct radiation through louvers in combination with heat pipes or an all heat pipe system. The propellant storage and feed system and thruster gimbal system concepts are presented. The three approaches are compared on the basis of mass, cost, testing, interfaces, simplicity, reliability, and maintainability.

Modular thrust subsystem approaches to solar electric propulsion module design

NASA Technical Reports Server (NTRS)

Cake, J. E.; Sharp, G. R.; Oglebay, J. C.; Shaker, F. J.; Zevesky, R. J.

1976-01-01

Three approaches are presented for packaging the elements of a 30 cm ion thrustor subsystem into a modular thrust subsystem. The individual modules, when integrated into a conceptual solar electric propulsion module are applicable to a multimission set of interplanetary flights with the Space Shuttle/Interim Upper Stage as the launch vehicle. The emphasis is on the structural and thermal integration of the components into the modular thrust subsystems. Thermal control for the power processing units is either by direct radiation through louvers in combination with heat pipes of an all heat pipe system. The propellant storage and feed system and thrustor gimbal system concepts are presented. The three approaches are compared on the basis of mass, cost, testing, interfaces, simplicity, reliability, and maintainability.

The CMS Data Management System

NASA Astrophysics Data System (ADS)

Giffels, M.; Guo, Y.; Kuznetsov, V.; Magini, N.; Wildish, T.

2014-06-01

The data management elements in CMS are scalable, modular, and designed to work together. The main components are PhEDEx, the data transfer and location system; the Data Booking Service (DBS), a metadata catalog; and the Data Aggregation Service (DAS), designed to aggregate views and provide them to users and services. Tens of thousands of samples have been cataloged and petabytes of data have been moved since the run began. The modular system has allowed the optimal use of appropriate underlying technologies. In this contribution we will discuss the use of both Oracle and NoSQL databases to implement the data management elements as well as the individual architectures chosen. We will discuss how the data management system functioned during the first run, and what improvements are planned in preparation for 2015.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

PubMed

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Myostatin-2 gene structure and polymorphism of the promoter and first intron in the marine fish Sparus aurata: evidence for DNA duplications and/or translocations.

PubMed

Nadjar-Boger, Elisabeth; Funkenstein, Bruria

2011-02-01

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. Fish express at least two genes for MSTN: MSTN-1 and MSTN-2. To date, MSTN-2 promoters have been cloned only from salmonids and zebrafish. Here we described the cloning and sequence analysis of MSTN-2 gene and its 5' flanking region in the marine fish Sparus aurata (saMSTN-2). We demonstrate the existence of three alleles of the promoter and three alleles of the first intron. Sequence comparison of the promoter region in the three alleles revealed that although the sequences of the first 1050 bp upstream of the translation start site are almost identical in the three alleles, a substantial sequence divergence is seen further upstream. Careful sequence analysis of the region upstream of the first 1050 bp in the three alleles identified several elements that appear to be repeated in some or all sequences, at different positions. This suggests that the promoter region of saMSTN-2 has been subjected to various chromosomal rearrangements during the course of evolution, reflecting either insertion or deletion events. Screening of several genomic DNA collections indicated differences in allele frequency, with allele 'b' being the most abundant, followed by allele 'c', whereas allele 'a' is relatively rare. Sequence analysis of saMSTN-2 gene also revealed polymorphism in the first intron, identifying three alleles. The length difference in alleles '1R' and '2R' of the first intron is due to the presence of one or two copies of a repeated block of approximately 150 bp, located at the 5' end of the first intron. The third allele, '4R', has an additional insertion of 323 bp located 116 bp upstream of the 3' end of the first intron. Analysis of several DNA collections showed that the '2R' allele is the most common, followed by the '4R' allele, whereas the '1R' allele is relatively rare. Progeny analysis of a full-sib family showed a Mendelian mode of inheritance of the two genetic loci. No clear association was found between the two genetic markers and growth rate. These results show for the first time a substantial degree of polymorphism in both the promoter and first intron of MSTN-2 gene in a perciform fish species which points to chromosomal rearrangements that took place during evolution.

Modular Exhaust Design and Manufacturing Techniques for Low Cost Mid Volume Rapid Buidl to Order Systems

DTIC Science & Technology

2014-08-06

the pressure field is uniform across them, but which allow mass flow to be diverted. Series elements have a constant mass flow across the ports...they can be used to calculate the pressure and mass flow after the element from the pressure and mass flow prior to the element, as shown in...the matrix product of each transfer matrix in turn. The final matrix gives no information about the pressures and mass flows within the element

Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding.

PubMed

Schuster, Astrid; Lopez, Jose V; Becking, Leontine E; Kelly, Michelle; Pomponi, Shirley A; Wörheide, Gert; Erpenbeck, Dirk; Cárdenas, Paco

2017-03-20

Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns. For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated. This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested.

Phylogenetics and Gene Structure Dynamics of Polygalacturonase Genes in Aspergillus and Neurospora crassa

PubMed Central

Hong, Jin-Sung; Ryu, Ki-Hyun; Kwon, Soon-Jae; Kim, Jin-Won; Kim, Kwang-Soo; Park, Kyong-Cheul

2013-01-01

Polygalacturonase (PG) gene is a typical gene family present in eukaryotes. Forty-nine PGs were mined from the genomes of Neurospora crassa and five Aspergillus species. The PGs were classified into 3 clades such as clade 1 for rhamno-PGs, clade 2 for exo-PGs and clade 3 for exo- and endo-PGs, which were further grouped into 13 sub-clades based on the polypeptide sequence similarity. In gene structure analysis, a total of 124 introns were present in 44 genes and five genes lacked introns to give an average of 2.5 introns per gene. Intron phase distribution was 64.5% for phase 0, 21.8% for phase 1, and 13.7% for phase 2, respectively. The introns varied in their sequences and their lengths ranged from 20 bp to 424 bp with an average of 65.9 bp, which is approximately half the size of introns in other fungal genes. There were 29 homologous intron blocks and 26 of those were sub-clade specific. Intron losses were counted in 18 introns in which no obvious phase preference for intron loss was observed. Eighteen introns were placed at novel positions, which is considerably higher than those of plant PGs. In an evolutionary sense both intron loss and gain must have taken place for shaping the current PGs in these fungi. Together with the small intron size, low conservation of homologous intron blocks and higher number of novel introns, PGs of fungal species seem to have recently undergone highly dynamic evolution. PMID:25288950

Phylogenetic Distribution of Intron Positions in Alpha-Amylase Genes of Bilateria Suggests Numerous Gains and Losses

PubMed Central

Da Lage, Jean-Luc; Maczkowiak, Frédérique; Cariou, Marie-Louise

2011-01-01

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that “resets” of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures. PMID:21611157

Transcription regulation by distal enhancers: who's in the loop?

PubMed

Stadhouders, Ralph; van den Heuvel, Anita; Kolovos, Petros; Jorna, Ruud; Leslie, Kris; Grosveld, Frank; Soler, Eric

2012-01-01

Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system.

An Element-Based Concurrent Partitioner for Unstructured Finite Element Meshes

NASA Technical Reports Server (NTRS)

Ding, Hong Q.; Ferraro, Robert D.

1996-01-01

A concurrent partitioner for partitioning unstructured finite element meshes on distributed memory architectures is developed. The partitioner uses an element-based partitioning strategy. Its main advantage over the more conventional node-based partitioning strategy is its modular programming approach to the development of parallel applications. The partitioner first partitions element centroids using a recursive inertial bisection algorithm. Elements and nodes then migrate according to the partitioned centroids, using a data request communication template for unpredictable incoming messages. Our scalable implementation is contrasted to a non-scalable implementation which is a straightforward parallelization of a sequential partitioner.

Influence of intron length on interaction characters between post-spliced intron and its CDS in ribosomal protein genes

NASA Astrophysics Data System (ADS)

Zhao, Xiaoqing; Li, Hong; Bao, Tonglaga; Ying, Zhiqiang

2012-09-01

Many experiment evidences showed that sequence structures of introns and intron loss/gain can influence gene expression, but current mechanisms did not refer to the functions of post-spliced introns directly. We propose that postspliced introns play their functions in gene expression by interacting with their mRNA sequences and the interaction is characterized by the matched segments between introns and their CDS. In this study, we investigated the interaction characters with length series by improved Smith-Waterman local alignment software for the ribosomal protein genes in C. elegans and D. melanogaster. Our results showed that RF values of five intron groups are significantly high in the central non-conserved region and very low in 5'-end and 3'-end splicing region. It is interesting that the number of the optimal matched regions gradually increases with intron length. Distributions of the optimal matched regions are different for five intron groups. Our study revealed that there are more interaction regions between longer introns and their CDS than shorter, and it provides a positive pattern for regulating the gene expression.

Intron-loss evolution of hatching enzyme genes in Teleostei

PubMed Central

2010-01-01

Background Hatching enzyme, belonging to the astacin metallo-protease family, digests egg envelope at embryo hatching. Orthologous genes of the enzyme are found in all vertebrate genomes. Recently, we found that exon-intron structures of the genes were conserved among tetrapods, while the genes of teleosts frequently lost their introns. Occurrence of such intron losses in teleostean hatching enzyme genes is an uncommon evolutionary event, as most eukaryotic genes are generally known to be interrupted by introns and the intron insertion sites are conserved from species to species. Here, we report on extensive studies of the exon-intron structures of teleostean hatching enzyme genes for insight into how and why introns were lost during evolution. Results We investigated the evolutionary pathway of intron-losses in hatching enzyme genes of 27 species of Teleostei. Hatching enzyme genes of basal teleosts are of only one type, which conserves the 9-exon-8-intron structure of an assumed ancestor. On the other hand, otocephalans and euteleosts possess two types of hatching enzyme genes, suggesting a gene duplication event in the common ancestor of otocephalans and euteleosts. The duplicated genes were classified into two clades, clades I and II, based on phylogenetic analysis. In otocephalans and euteleosts, clade I genes developed a phylogeny-specific structure, such as an 8-exon-7-intron, 5-exon-4-intron, 4-exon-3-intron or intron-less structure. In contrast to the clade I genes, the structures of clade II genes were relatively stable in their configuration, and were similar to that of the ancestral genes. Expression analyses revealed that hatching enzyme genes were high-expression genes, when compared to that of housekeeping genes. When expression levels were compared between clade I and II genes, clade I genes tends to be expressed more highly than clade II genes. Conclusions Hatching enzyme genes evolved to lose their introns, and the intron-loss events occurred at the specific points of teleostean phylogeny. We propose that the high-expression hatching enzyme genes frequently lost their introns during the evolution of teleosts, while the low-expression genes maintained the exon-intron structure of the ancestral gene. PMID:20796321

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1.

PubMed

Wong, Jing Ting; Akhbar, Farzanah; Ng, Amanda Yunn Ee; Tay, Mandy Li-Ian; Loi, Gladys Jing En; Pek, Jun Wei

2017-10-02

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. Here, the authors show that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis.

Insertion of an SVA-E retrotransposon into the CASP8 gene is associated with protection against prostate cancer

PubMed Central

Stacey, Simon N.; Kehr, Birte; Gudmundsson, Julius; Zink, Florian; Jonasdottir, Aslaug; Gudjonsson, Sigurjon A.; Sigurdsson, Asgeir; Halldorsson, Bjarni V.; Agnarsson, Bjarni A.; Benediktsdottir, Kristrun R.; Aben, Katja K.H.; Vermeulen, Sita H.; Cremers, Ruben G.; Panadero, Angeles; Helfand, Brian T.; Cooper, Phillip R.; Donovan, Jenny L.; Hamdy, Freddie C.; Jinga, Viorel; Okamoto, Ichiro; Jonasson, Jon G.; Tryggvadottir, Laufey; Johannsdottir, Hrefna; Kristinsdottir, Anna M.; Masson, Gisli; Magnusson, Olafur T.; Iordache, Paul D.; Helgason, Agnar; Helgason, Hannes; Sulem, Patrick; Gudbjartsson, Daniel F.; Kong, Augustine; Jonsson, Eirikur; Barkardottir, Rosa B.; Einarsson, Gudmundur V.; Rafnar, Thorunn; Thorsteinsdottir, Unnur; Mates, Ioan N.; Neal, David E.; Catalona, William J.; Mayordomo, José I.; Kiemeney, Lambertus A.; Thorleifsson, Gudmar; Stefansson, Kari

2016-01-01

Transcriptional and splicing anomalies have been observed in intron 8 of the CASP8 gene (encoding procaspase-8) in association with cutaneous basal-cell carcinoma (BCC) and linked to a germline SNP rs700635. Here, we show that the rs700635[C] allele, which is associated with increased risk of BCC and breast cancer, is protective against prostate cancer [odds ratio (OR) = 0.91, P = 1.0 × 10−6]. rs700635[C] is also associated with failures to correctly splice out CASP8 intron 8 in breast and prostate tumours and in corresponding normal tissues. Investigation of rs700635[C] carriers revealed that they have a human-specific short interspersed element-variable number of tandem repeat-Alu (SINE-VNTR-Alu), subfamily-E retrotransposon (SVA-E) inserted into CASP8 intron 8. The SVA-E shows evidence of prior activity, because it has transduced some CASP8 sequences during subsequent retrotransposition events. Whole-genome sequence (WGS) data were used to tag the SVA-E with a surrogate SNP rs1035142[T] (r2 = 0.999), which showed associations with both the splicing anomalies (P = 6.5 × 10−32) and with protection against prostate cancer (OR = 0.91, P = 3.8 × 10−7). PMID:26740556

An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii.

PubMed

Berthold, Peter; Schmitt, Rüdiger; Mages, Wolfgang

2002-12-01

We have developed a positively selectable marker for the green alga Chlamydomonas reinhardtii using the Streptomyces hygroscopicus aminoglycoside phosphotransferase gene (aph7"). Its expression is controlled by C. reinhardtii regulatory elements, namely, the beta2-tubulin gene promoter in combination with the first intron and the 3' untranslated region of the small subunit of ribulose bisphosphate carboxylase, rbcS2. C. reinhardtii cell-wall deficient and wild-type strains were transformed at rates up to 5 x 10(-5) with two constructs, pHyg3 and pHyg4 (intron-less). Transformants selected on plates with 10 microg/ml hygromycin B exhibited diverse levels of resistance of up to 200 microg/ml that were stably maintained for at least seven months; they contained two to five copies of the construct integrated in their genomes. Transcription of the chimeric aph7" gene, correct splicing of the rbcS2 intron, and polyadenylation of the transcripts have been verified by sequencing of RT-PCR products. Average co-transformation rates using pHyg3 and a second selectable plasmid were about 11%. This advocates the hygromycin-resistance plasmid, pHyg3, as a new versatile tool for the transformation of a broad range of C. reinhardtii strains without the sustained need for using auxotrophic mutants as recipients.

High variability of mitochondrial gene order among fungi.

PubMed

Aguileta, Gabriela; de Vienne, Damien M; Ross, Oliver N; Hood, Michael E; Giraud, Tatiana; Petit, Elsa; Gabaldón, Toni

2014-02-01

From their origin as an early alpha proteobacterial endosymbiont to their current state as cellular organelles, large-scale genomic reorganization has taken place in the mitochondria of all main eukaryotic lineages. So far, most studies have focused on plant and animal mitochondrial (mt) genomes (mtDNA), but fungi provide new opportunities to study highly differentiated mtDNAs. Here, we analyzed 38 complete fungal mt genomes to investigate the evolution of mtDNA gene order among fungi. In particular, we looked for evidence of nonhomologous intrachromosomal recombination and investigated the dynamics of gene rearrangements. We investigated the effect that introns, intronic open reading frames (ORFs), and repeats may have on gene order. Additionally, we asked whether the distribution of transfer RNAs (tRNAs) evolves independently to that of mt protein-coding genes. We found that fungal mt genomes display remarkable variation between and within the major fungal phyla in terms of gene order, genome size, composition of intergenic regions, and presence of repeats, introns, and associated ORFs. Our results support previous evidence for the presence of mt recombination in all fungal phyla, a process conspicuously lacking in most Metazoa. Overall, the patterns of rearrangements may be explained by the combined influences of recombination (i.e., most likely nonhomologous and intrachromosomal), accumulated repeats, especially at intergenic regions, and to a lesser extent, mobile element dynamics.

Remarkable sequence conservation of the last intron in the PKD1 gene.

PubMed

Rodova, Marianna; Islam, M Rafiq; Peterson, Kenneth R; Calvet, James P

2003-10-01

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Structural, functional and evolutionary relationships between homing endonucleases and proteins from their host organisms

PubMed Central

Taylor, Gregory K.; Stoddard, Barry L.

2012-01-01

Homing endonucleases (HEs) are highly specific DNA-cleaving enzymes that are encoded by invasive DNA elements (usually mobile introns or inteins) within the genomes of phage, bacteria, archea, protista and eukaryotic organelles. Six unique structural HE families, that collectively span four distinct nuclease catalytic motifs, have been characterized to date. Members of each family display structural homology and functional relationships to a wide variety of proteins from various organisms. The biological functions of those proteins are highly disparate and include non-specific DNA-degradation enzymes, restriction endonucleases, DNA-repair enzymes, resolvases, intron splicing factors and transcription factors. These relationships suggest that modern day HEs share common ancestors with proteins involved in genome fidelity, maintenance and gene expression. This review summarizes the results of structural studies of HEs and corresponding proteins from host organisms that have illustrated the manner in which these factors are related. PMID:22406833

Introns in Cryptococcus.

PubMed

Janbon, Guilhem

2018-01-01

In Cryptococcus neoformans, nearly all genes are interrupted by small introns. In recent years, genome annotation and genetic analysis have illuminated the major roles these introns play in the biology of this pathogenic yeast. Introns are necessary for gene expression and alternative splicing can regulate gene expression in response to environmental cues. In addition, recent studies have revealed that C. neoformans introns help to prevent transposon dissemination and protect genome integrity. These characteristics of cryptococcal introns are probably not unique to Cryptococcus, and this yeast likely can be considered as a model for intron-related studies in fungi.

DNA double-strand break in vivo at the 3' extremity of exons located upstream of group II introns. Senescence and circular DNA introns in Podospora mitochondria.

PubMed

Sainsard-Chanet, A; Begel, O; Belcour, L

1994-10-07

In the filamentous fungus Podospora anserina, the unavoidable phenomenon of senescence is associated with the amplification of the first intron of the mitochondrial cox1 that accumulates as circular DNA molecules consisting of tandem repeats. This group II intron (cox1-i1 or alpha) is able to transpose and contains an open reading frame with significant amino acid similarity with reverse transcriptases. The generation of these intronic circular DNA molecules, their amplification and their involvement in the senescence process are unresolved questions. We demonstrate here that: (1) another group II intron, the fourth intron of gene cox1, cox1-i4, is also able to give precise DNA end to end junctions; (2) this intronic sequence can be found amplified during senescence, although to a lesser extent than cox1-i1; (3) the amplification of the DNA multimeric cox1-i1 molecules likely does not proceed by autonomous replication; (4) the generation of the DNA intronic circles does not require efficient intron splicing; (5) a DNA double-strand break occurs in vivo at the 3' extremity of the cox1-e1 and cox1-e4 exons preceding the group II introns that form circular DNAs. On the whole, these results show that the ability to form DNA circular molecules is a property of some group II introns and they demonstrate the occurrence of a specific DNA cleavage at or near the integration site of these group II introns. The results strongly suggest that this cleavage is involved in the formation of the group II intronic DNA circles and could also be involved in the phenomenon of group II intron homing.

Branchpoint selection in the splicing of U12-dependent introns in vitro.

PubMed

McConnell, Timothy S; Cho, Soo-Jin; Frilander, Mikko J; Steitz, Joan A

2002-05-01

In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome.

Branchpoint selection in the splicing of U12-dependent introns in vitro.

PubMed Central

McConnell, Timothy S; Cho, Soo-Jin; Frilander, Mikko J; Steitz, Joan A

2002-01-01

In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome. PMID:12022225

Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.

PubMed

Ishida, Ken; Kuboshima, Megumi; Morita, Hiroto; Maeda, Hiroshi; Okamoto, Ayako; Takeuchi, Michio; Yamagata, Youhei

2014-01-01

Alternative splicing is thought to be a means for diversification of products by mRNA modification. Although some intron retentions are predicted by transcriptome analysis in Aspergillus oryzae, its physiological significance remains unknown. We found that intron retention occurred occasionally in the serine-type carboxypeptidase gene, ocpG. Analysis under various culture conditions revealed that extracellular nitrogen conditions influence splicing patterns; this suggested that there might be a correlation between splicing efficiency and the necessity of OcpG activity for obtaining a nitrogen source. Since further analysis showed that splicing occurred independently in each intron, we constructed ocpG intron-exchanging strain by interchanging the positions of intron-1 and intron-2. The splicing pattern indicated the probability that ocpG intron retention was affected by the secondary structures of intronic mRNA.

A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution.

PubMed Central

Copertino, D W; Christopher, D A; Hallick, R B

1991-01-01

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts. Images PMID:1721702

Defective neuronal migration and inhibition of bipolar to multipolar transition of migrating neural cells by Mesoderm-Specific Transcript, Mest, in the developing mouse neocortex.

PubMed

Ji, Liting; Bishayee, Kausik; Sadra, Ali; Choi, Seunghyuk; Choi, Wooyul; Moon, Sungho; Jho, Eek-Hoon; Huh, Sung-Oh

2017-07-04

Brain developmental disorders such as lissencephaly can result from faulty neuronal migration and differentiation during the formation of the mammalian neocortex. The cerebral cortex is a modular structure, where developmentally, newborn neurons are generated as a neuro-epithelial sheet and subsequently differentiate, migrate and organize into their final positions in the cerebral cortical plate via a process involving both tangential and radial migration. The specific role of Mest, an imprinted gene, in neuronal migration has not been previously studied. In this work, we reduced expression of Mest with in utero electroporation of neuronal progenitors in the developing embryonic mouse neocortex. Reduction of Mest levels by shRNA significantly reduced the number of neurons migrating to the cortical plate. Also, Mest-knockdown disrupted the transition of bipolar neurons into multipolar neurons migrating out of the sub-ventricular zone region. The migrating neurons also adopted a more tangential migration pattern upon knockdown of the Mest message, losing their potential to attach to radial glia cells, required for radial migration. The differentiation and migration properties of neurons via Wnt-Akt signaling were affected by Mest changes. In addition, miR-335, encoded in a Mest gene intron, was identified as being responsible for blocking the default tangential migration of the neurons. Our results suggest that Mest and its intron product, miR-335, play important roles in neuronal migration with Mest regulating the morphological transition of primary neurons required in the formation of the mammalian neocortex. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence.

PubMed

Akkuratov, Evgeny E; Walters, Lorraine; Saha-Mandal, Arnab; Khandekar, Sushant; Crawford, Erin; Zirbel, Craig L; Leisner, Scott; Prakash, Ashwin; Fedorova, Larisa; Fedorov, Alexei

2014-09-10

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection

PubMed Central

Frazier, Courtney L.; San Filippo, Joseph; Lambowitz, Alan M.; Mills, David A.

2003-01-01

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci. PMID:12571038

Evolution of introns in the archaeal world.

PubMed

Tocchini-Valentini, Giuseppe D; Fruscoloni, Paolo; Tocchini-Valentini, Glauco P

2011-03-22

The self-splicing group I introns are removed by an autocatalytic mechanism that involves a series of transesterification reactions. They require RNA binding proteins to act as chaperones to correctly fold the RNA into an active intermediate structure in vivo. Pre-tRNA introns in Bacteria and in higher eukaryote plastids are typical examples of self-splicing group I introns. By contrast, two striking features characterize RNA splicing in the archaeal world. First, self-splicing group I introns cannot be found, to this date, in that kingdom. Second, the RNA splicing scenario in Archaea is uniform: All introns, whether in pre-tRNA or elsewhere, are removed by tRNA splicing endonucleases. We suggest that in Archaea, the protein recruited for splicing is the preexisting tRNA splicing endonuclease and that this enzyme, together with the ligase, takes over the task of intron removal in a more efficient fashion than the ribozyme. The extinction of group I introns in Archaea would then be a consequence of recruitment of the tRNA splicing endonuclease. We deal here with comparative genome analysis, focusing specifically on the integration of introns into genes coding for 23S rRNA molecules, and how this newly acquired intron has to be removed to regenerate a functional RNA molecule. We show that all known oligomeric structures of the endonuclease can recognize and cleave a ribosomal intron, even when the endonuclease derives from a strain lacking rRNA introns. The persistence of group I introns in mitochondria and chloroplasts would be explained by the inaccessibility of these introns to the endonuclease.

Hypervariable and highly divergent intron-exon organizations in the chordate Oikopleura dioica.

PubMed

Edvardsen, Rolf B; Lerat, Emmanuelle; Maeland, Anne Dorthea; Flåt, Mette; Tewari, Rita; Jensen, Marit F; Lehrach, Hans; Reinhardt, Richard; Seo, Hee-Chan; Chourrout, Daniel

2004-10-01

Oikopleura dioica is a pelagic tunicate with a very small genome and a very short life cycle. In order to investigate the intron-exon organizations in Oikopleura, we have isolated and characterized ribosomal protein EF-1alpha, Hox, and alpha-tubulin genes. Their intron positions have been compared with those of the same genes from various invertebrates and vertebrates, including four species with entirely sequenced genomes. Oikopleura genes, like Caenorhabditis genes, have introns at a large number of nonconserved positions, which must originate from late insertions or intron sliding of ancient insertions. Both species exhibit hypervariable intron-exon organization within their alpha-tubulin gene family. This is due to localization of most nonconserved intron positions in single members of this gene family. The hypervariability and divergence of intron positions in Oikopleura and Caenorhabditis may be related to the predominance of short introns, the processing of which is not very dependent upon the exonic environment compared to large introns. Also, both species have an undermethylated genome, and the control of methylation-induced point mutations imposes a control on exon size, at least in vertebrate genes. That introns placed at such variable positions in Oikopleura or C. elegans may serve a specific purpose is not easy to infer from our current knowledge and hypotheses on intron functions. We propose that new introns are retained in species with very short life cycles, because illegitimate exchanges including gene conversion are repressed. We also speculate that introns placed at gene-specific positions may contribute to suppressing these exchanges and thereby favor their own persistence.

The Mitochondrial Genome of the Prasinophyte Prasinoderma coloniale Reveals Two Trans-Spliced Group I Introns in the Large Subunit rRNA Gene

PubMed Central

Pombert, Jean-François; Otis, Christian; Turmel, Monique; Lemieux, Claude

2013-01-01

Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications of this interesting observation for trans-splicing of group I introns. PMID:24386369

Scalable Light Module for Low-Cost, High-Efficiency Light- Emitting Diode Luminaires

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

Tarsa, Eric

2015-08-31

During this two-year program Cree developed a scalable, modular optical architecture for low-cost, high-efficacy light emitting diode (LED) luminaires. Stated simply, the goal of this architecture was to efficiently and cost-effectively convey light from LEDs (point sources) to broad luminaire surfaces (area sources). By simultaneously developing warm-white LED components and low-cost, scalable optical elements, a high system optical efficiency resulted. To meet program goals, Cree evaluated novel approaches to improve LED component efficacy at high color quality while not sacrificing LED optical efficiency relative to conventional packages. Meanwhile, efficiently coupling light from LEDs into modular optical elements, followed by optimallymore » distributing and extracting this light, were challenges that were addressed via novel optical design coupled with frequent experimental evaluations. Minimizing luminaire bill of materials and assembly costs were two guiding principles for all design work, in the effort to achieve luminaires with significantly lower normalized cost ($/klm) than existing LED fixtures. Chief project accomplishments included the achievement of >150 lm/W warm-white LEDs having primary optics compatible with low-cost modular optical elements. In addition, a prototype Light Module optical efficiency of over 90% was measured, demonstrating the potential of this scalable architecture for ultra-high-efficacy LED luminaires. Since the project ended, Cree has continued to evaluate optical element fabrication and assembly methods in an effort to rapidly transfer this scalable, cost-effective technology to Cree production development groups. The Light Module concept is likely to make a strong contribution to the development of new cost-effective, high-efficacy luminaries, thereby accelerating widespread adoption of energy-saving SSL in the U.S.« 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.

Structure of a group II intron in complex with its reverse transcriptase.

PubMed

Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

2016-06-01

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

Ancient nature of alternative splicing and functions of introns

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

Zhou, Kemin; Salamov, Asaf; Kuo, Alan

Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but intronsmore » retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.« less

Remarkable interkingdom conservation of intron positions and massive, lineage-specific intron loss and gain in eukaryotic evolution.

PubMed

Rogozin, Igor B; Wolf, Yuri I; Sorokin, Alexander V; Mirkin, Boris G; Koonin, Eugene V

2003-09-02

Sequencing of eukaryotic genomes allows one to address major evolutionary problems, such as the evolution of gene structure. We compared the intron positions in 684 orthologous gene sets from 8 complete genomes of animals, plants, fungi, and protists and constructed parsimonious scenarios of evolution of the exon-intron structure for the respective genes. Approximately one-third of the introns in the malaria parasite Plasmodium falciparum are shared with at least one crown group eukaryote; this number indicates that these introns have been conserved through >1.5 billion years of evolution that separate Plasmodium from the crown group. Paradoxically, humans share many more introns with the plant Arabidopsis thaliana than with the fly or nematode. The inferred evolutionary scenario holds that the common ancestor of Plasmodium and the crown group and, especially, the common ancestor of animals, plants, and fungi had numerous introns. Most of these ancestral introns, which are retained in the genomes of vertebrates and plants, have been lost in fungi, nematodes, arthropods, and probably Plasmodium. In addition, numerous introns have been inserted into vertebrate and plant genes, whereas, in other lineages, intron gain was much less prominent.

A Detailed History of Intron-rich Eukaryotic Ancestors Inferred from a Global Survey of 100 Complete Genomes

PubMed Central

Csuros, Miklos; Rogozin, Igor B.; Koonin, Eugene V.

2011-01-01

Protein-coding genes in eukaryotes are interrupted by introns, but intron densities widely differ between eukaryotic lineages. Vertebrates, some invertebrates and green plants have intron-rich genes, with 6–7 introns per kilobase of coding sequence, whereas most of the other eukaryotes have intron-poor genes. We reconstructed the history of intron gain and loss using a probabilistic Markov model (Markov Chain Monte Carlo, MCMC) on 245 orthologous genes from 99 genomes representing the three of the five supergroups of eukaryotes for which multiple genome sequences are available. Intron-rich ancestors are confidently reconstructed for each major group, with 53 to 74% of the human intron density inferred with 95% confidence for the Last Eukaryotic Common Ancestor (LECA). The results of the MCMC reconstruction are compared with the reconstructions obtained using Maximum Likelihood (ML) and Dollo parsimony methods. An excellent agreement between the MCMC and ML inferences is demonstrated whereas Dollo parsimony introduces a noticeable bias in the estimations, typically yielding lower ancestral intron densities than MCMC and ML. Evolution of eukaryotic genes was dominated by intron loss, with substantial gain only at the bases of several major branches including plants and animals. The highest intron density, 120 to 130% of the human value, is inferred for the last common ancestor of animals. The reconstruction shows that the entire line of descent from LECA to mammals was intron-rich, a state conducive to the evolution of alternative splicing. PMID:21935348

Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria.

PubMed

Rot, Chagai; Goldfarb, Itay; Ilan, Micha; Huchon, Dorothée

2006-09-14

The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 and nad5 genes. Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera). A 2,349 bp fragment of the mitochondrial cox1 gene was sequenced from the sponge Tetilla sp. (Spirophorida). This fragment suggests the presence of a 1143 bp intron. Similar to all the cnidarian mitochondrial introns, the putative intron has group I intron characteristics. The intron is present in the cox1 gene and encodes a putative homing endonuclease. In order to establish the distribution of this intron in sponges, the cox1 gene was sequenced from several representatives of the demosponge diversity. The intron was found only in the sponge order Spirophorida. A phylogenetic analysis of the COI protein sequence and of the intron open reading frame suggests that the intron may have been transmitted horizontally from a fungus donor. Little is known about sponge-associated fungi, although in the last few years the latter have been frequently isolated from sponges. We suggest that the horizontal gene transfer of a mitochondrial intron was facilitated by a symbiotic relationship between fungus and sponge. Ecological relationships are known to have implications at the genomic level. Here, an ecological relationship between sponge and fungus is suggested based on the genomic analysis.

Spectral statistics of the uni-modular ensemble

NASA Astrophysics Data System (ADS)

Joyner, Christopher H.; Smilansky, Uzy; Weidenmüller, Hans A.

2017-09-01

We investigate the spectral statistics of Hermitian matrices in which the elements are chosen uniformly from U(1) , called the uni-modular ensemble (UME), in the limit of large matrix size. Using three complimentary methods; a supersymmetric integration method, a combinatorial graph-theoretical analysis and a Brownian motion approach, we are able to derive expressions for 1 / N corrections to the mean spectral moments and also analyse the fluctuations about this mean. By addressing the same ensemble from three different point of view, we can critically compare their relative advantages and derive some new results.

Germline EMSY sequence alterations in hereditary breast cancer and ovarian cancer families.

PubMed

Määttä, Kirsi M; Nurminen, Riikka; Kankuri-Tammilehto, Minna; Kallioniemi, Anne; Laasanen, Satu-Leena; Schleutker, Johanna

2017-07-24

BRCA1 and BRCA2 mutations explain approximately one-fifth of the inherited susceptibility in high-risk Finnish hereditary breast and ovarian cancer (HBOC) families. EMSY is located in the breast cancer-associated chromosomal region 11q13. The EMSY gene encodes a BRCA2-interacting protein that has been implicated in DNA damage repair and genomic instability. We analysed the role of germline EMSY variation in breast/ovarian cancer predisposition. The present study describes the first EMSY screening in patients with high familial risk for this disease. Index individuals from 71 high-risk, BRCA1/2-negative HBOC families were screened for germline EMSY sequence alterations in protein coding regions and exon-intron boundaries using Sanger sequencing and TaqMan assays. The identified variants were further screened in 36 Finnish HBOC patients and 904 controls. Moreover, one novel intronic deletion was screened in a cohort of 404 breast cancer patients unselected for family history. Haplotype block structure and the association of haplotypes with breast/ovarian cancer were analysed using Haploview. The functionality of the identified variants was predicted using Haploreg, RegulomeDB, Human Splicing Finder, and Pathogenic-or-Not-Pipeline 2. Altogether, 12 germline EMSY variants were observed. Two alterations were located in the coding region, five alterations were intronic, and five alterations were located in the 3'untranslated region (UTR). Variant frequencies did not significantly differ between cases and controls. The novel variant, c.2709 + 122delT, was detected in 1 out of 107 (0.9%) breast cancer patients, and the carrier showed a bilateral form of the disease. The deletion was absent in 897 controls (OR = 25.28; P = 0.1) and in 404 breast cancer patients unselected for family history. No haplotype was identified to increase the risk of breast/ovarian cancer. Functional analyses suggested that variants, particularly in the 3'UTR, were located within regulatory elements. The novel deletion was predicted to affect splicing regulatory elements. These results suggest that the identified EMSY variants are likely neutral at the population level. However, these variants may contribute to breast/ovarian cancer risk in single families. Additional analyses are warranted for rare novel intronic deletions and the 3'UTR variants predicted to have functional roles.

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster

PubMed Central

Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S.; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

2014-01-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as ‘homing’ similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. PMID:24803674

Functional autonomy of distant-acting human enhancers

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

Visel, Axel; Akiyama, Jennifer A.; Shoukry, Malak

2009-02-19

Many human genes are associated with dispersed arrays of transcriptional enhancers that regulate their expression in time and space. Studies in invertebrate model systems have suggested that these elements function as discrete and independent regulatory units, but the in vivo combinatorial properties of vertebrate enhancers remain poorly understood. To explore the modularity and regulatory autonomy of human developmental enhancers, we experimentally concatenated up to four enhancers from different genes and used a transgenic mouse assay to compare the in vivo activity of these compound elements with that of the single modules. In all of the six different combinations of elementsmore » tested, the reporter gene activity patterns were additive without signs of interference between the individual modules, indicating that regulatory specificity was maintained despite the presence of closely-positioned heterologous enhancers. Even in cases where two elements drove expression in close anatomical proximity, such as within neighboring subregions of the developing limb bud, the compound patterns did not show signs of cross-inhibition between individual elements or novel expression sites. These data indicate that human developmental enhancers are highly modular and functionally autonomous and suggest that genomic enhancer shuffling may have contributed to the evolution of complex gene expression patterns in vertebrates« less

Localized Retroprocessing as a Model of Intron Loss in the Plant Mitochondrial Genome

PubMed Central

Cuenca, Argelia; Ross, T. Gregory; Graham, Sean W.; Barrett, Craig F.; Davis, Jerrold I.; Seberg, Ole; Petersen, Gitte

2016-01-01

Loss of introns in plant mitochondrial genes is commonly explained by retroprocessing. Under this model, an mRNA is reverse transcribed and integrated back into the genome, simultaneously affecting the contents of introns and edited sites. To evaluate the extent to which retroprocessing explains intron loss, we analyzed patterns of intron content and predicted RNA editing for whole mitochondrial genomes of 30 species in the monocot order Alismatales. In this group, we found an unusually high degree of variation in the intron content, even expanding the hitherto known variation among angiosperms. Some species have lost some two-third of the cis-spliced introns. We found a strong correlation between intron content and editing frequency, and detected 27 events in which intron loss is consistent with the presence of nucleotides in an edited state, supporting retroprocessing. However, we also detected seven cases of intron loss not readily being explained by retroprocession. Our analyses are also not consistent with the entire length of a fully processed cDNA copy being integrated into the genome, but instead indicate that retroprocessing usually occurs for only part of the gene. In some cases, several rounds of retroprocessing may explain intron loss in genes completely devoid of introns. A number of taxa retroprocessing seem to be very common and a possibly ongoing process. It affects the entire mitochondrial genome. PMID:27435795

Euglena gracilis chloroplast DNA: analysis of a 1.6 kb intron of the psb C gene containing an open reading frame of 458 codons.

PubMed

Montandon, P E; Vasserot, A; Stutz, E

1986-01-01

We retrieved a 1.6 kbp intron separating two exons of the psb C gene which codes for the 44 kDa reaction center protein of photosystem II. This intron is 3 to 4 times the size of all previously sequenced Euglena gracilis chloroplast introns. It contains an open reading frame of 458 codons potentially coding for a basic protein of 54 kDa of yet unknown function. The intron boundaries follow consensus sequences established for chloroplast introns related to class II and nuclear pre-mRNA introns. Its 3'-terminal segment has structural features similar to class II mitochondrial introns with an invariant base A as possible branch point for lariat formation.

[Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae].

PubMed

Kamenskaya, D N; Pankova, M V; Atopkin, D M; Brykov, V A

2017-01-01

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

Epigenetic regulation of intragenic transposable elements impacts gene transcription in Arabidopsis thaliana

PubMed Central

Le, Tu N.; Miyazaki, Yuji; Takuno, Shohei; Saze, Hidetoshi

2015-01-01

Genomes of higher eukaryotes, including plants, contain numerous transposable elements (TEs), that are often silenced by epigenetic mechanisms, such as histone modifications and DNA methylation. Although TE silencing adversely affects expression of nearby genes, recent studies reveal the presence of intragenic TEs marked by repressive heterochromatic epigenetic marks within transcribed genes. However, even for the well-studied plant model Arabidopsis thaliana, the abundance of intragenic TEs, how they are epigenetically regulated, and their potential impacts on host gene expression, remain unexplored. In this study, we comprehensively analyzed genome-wide distribution and epigenetic regulation of intragenic TEs in A. thaliana. Our analysis revealed that about 3% of TEs are located within gene bodies, dominantly at intronic regions. Most of them are shorter and less methylated than intergenic TEs, but they are still targeted by RNA-directed DNA methylation-dependent and independent pathways. Surprisingly, the heterochromatic epigenetic marks at TEs are maintained within actively transcribed genes. Moreover, the heterochromatic state of intronic TEs is critical for proper transcription of associated genes. Our study provides the first insight into how intragenic TEs affect the transcriptional landscape of the A. thaliana genome, and suggests the importance of epigenetic mechanisms for regulation of TEs within transcriptional gene units. PMID:25813042

Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria

PubMed Central

Rot, Chagai; Goldfarb, Itay; Ilan, Micha; Huchon, Dorothée

2006-01-01

Background The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 and nad5 genes. Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera). Results A 2,349 bp fragment of the mitochondrial cox1 gene was sequenced from the sponge Tetilla sp. (Spirophorida). This fragment suggests the presence of a 1143 bp intron. Similar to all the cnidarian mitochondrial introns, the putative intron has group I intron characteristics. The intron is present in the cox1 gene and encodes a putative homing endonuclease. In order to establish the distribution of this intron in sponges, the cox1 gene was sequenced from several representatives of the demosponge diversity. The intron was found only in the sponge order Spirophorida. A phylogenetic analysis of the COI protein sequence and of the intron open reading frame suggests that the intron may have been transmitted horizontally from a fungus donor. Conclusion Little is known about sponge-associated fungi, although in the last few years the latter have been frequently isolated from sponges. We suggest that the horizontal gene transfer of a mitochondrial intron was facilitated by a symbiotic relationship between fungus and sponge. Ecological relationships are known to have implications at the genomic level. Here, an ecological relationship between sponge and fungus is suggested based on the genomic analysis. PMID:16972986

Exon definition as a potential negative force against intron losses in evolution.

PubMed

Niu, Deng-Ke

2008-11-13

Previous studies have indicated that the wide variation in intron density (the number of introns per gene) among different eukaryotes largely reflects varying degrees of intron loss during evolution. The most popular model, which suggests that organisms lose introns through a mechanism in which reverse-transcribed cDNA recombines with the genomic DNA, concerns only one mutational force. Using exons as the units of splicing-site recognition, exon definition constrains the length of exons. An intron-loss event results in fusion of flanking exons and thus a larger exon. The large size of the newborn exon may cause splicing errors, i.e., exon skipping, if the splicing of pre-mRNAs is initiated by exon definition. By contrast, if the splicing of pre-mRNAs is initiated by intron definition, intron loss does not matter. Exon definition may thus be a selective force against intron loss. An organism with a high frequency of exon definition is expected to experience a low rate of intron loss throughout evolution and have a high density of spliceosomal introns. The majority of spliceosomal introns in vertebrates may be maintained during evolution not because of potential functions, but because of their splicing mechanism (i.e., exon definition). Further research is required to determine whether exon definition is a negative force in maintaining the high intron density of vertebrates. This article was reviewed by Dr. Scott W. Roy (nominated by Dr. John Logsdon), Dr.Eugene V. Koonin, and Dr. Igor B. Rogozin (nominated by Dr. Mikhail Gelfand). For the full reviews,please go to the Reviewers' comments section.

Standardized Modular Power Interfaces for Future Space Explorations Missions

NASA Technical Reports Server (NTRS)

Oeftering, Richard

2015-01-01

Earlier studies show that future human explorations missions are composed of multi-vehicle assemblies with interconnected electric power systems. Some vehicles are often intended to serve as flexible multi-purpose or multi-mission platforms. This drives the need for power architectures that can be reconfigured to support this level of flexibility. Power system developmental costs can be reduced, program wide, by utilizing a common set of modular building blocks. Further, there are mission operational and logistics cost benefits of using a common set of modular spares. These benefits are the goals of the Advanced Exploration Systems (AES) Modular Power System (AMPS) project. A common set of modular blocks requires a substantial level of standardization in terms of the Electrical, Data System, and Mechanical interfaces. The AMPS project is developing a set of proposed interface standards that will provide useful guidance for modular hardware developers but not needlessly constrain technology options, or limit future growth in capability. In 2015 the AMPS project focused on standardizing the interfaces between the elements of spacecraft power distribution and energy storage. The development of the modular power standard starts with establishing mission assumptions and ground rules to define design application space. The standards are defined in terms of AMPS objectives including Commonality, Reliability-Availability, Flexibility-Configurability and Supportability-Reusability. The proposed standards are aimed at assembly and sub-assembly level building blocks. AMPS plans to adopt existing standards for spacecraft command and data, software, network interfaces, and electrical power interfaces where applicable. Other standards including structural encapsulation, heat transfer, and fluid transfer, are governed by launch and spacecraft environments and bound by practical limitations of weight and volume. Developing these mechanical interface standards is more difficult but an essential part of defining physical building blocks of modular power. This presentation describes the AMPS projects progress towards standardized modular power interfaces.

Semi-Infinite Geology Modeling Algorithm (SIGMA): a Modular Approach to 3D Gravity

NASA Astrophysics Data System (ADS)

Chang, J. C.; Crain, K.

2015-12-01

Conventional 3D gravity computations can take up to days, weeks, and even months, depending on the size and resolution of the data being modeled. Additional modeling runs, due to technical malfunctions or additional data modifications, only compound computation times even further. We propose a new modeling algorithm that utilizes vertical line elements to approximate mass, and non-gridded (point) gravity observations. This algorithm is (1) magnitudes faster than conventional methods, (2) accurate to less than 0.1% error, and (3) modular. The modularity of this methodology means that researchers can modify their geology/terrain or gravity data, and only the modified component needs to be re-run. Additionally, land-, sea-, and air-based platforms can be modeled at their observation point, without having to filter data into a synthesized grid.

Flat-panel video resolution LED display system

NASA Astrophysics Data System (ADS)

Wareberg, P. G.; Kennedy, D. I.

The system consists of a 128 x 128 element X-Y addressable LED array fabricated from green-emitting gallium phosphide. The LED array is interfaced with a 128 x 128 matrix TV camera. Associated electronics provides for seven levels of grey scale above zero with a grey scale ratio of square root of 2. Picture elements are on 0.008 inch centers resulting in a resolution of 125 lines-per-inch and a display area of approximately 1 sq. in. The LED array concept lends itself to modular construction, permitting assembly of a flat panel screen of any desired size from 1 x 1 inch building blocks without loss of resolution. A wide range of prospective aerospace applications exist extending from helmet-mounted systems involving small dedicated arrays to multimode cockpit displays constructed as modular screens. High-resolution LED arrays are already used as CRT replacements in military film-marking reconnaissance applications.

Computational Approaches to Identify Promoters and cis-Regulatory Elements in Plant Genomes1

PubMed Central

Rombauts, Stephane; Florquin, Kobe; Lescot, Magali; Marchal, Kathleen; Rouzé, Pierre; Van de Peer, Yves

2003-01-01

The identification of promoters and their regulatory elements is one of the major challenges in bioinformatics and integrates comparative, structural, and functional genomics. Many different approaches have been developed to detect conserved motifs in a set of genes that are either coregulated or orthologous. However, although recent approaches seem promising, in general, unambiguous identification of regulatory elements is not straightforward. The delineation of promoters is even harder, due to its complex nature, and in silico promoter prediction is still in its infancy. Here, we review the different approaches that have been developed for identifying promoters and their regulatory elements. We discuss the detection of cis-acting regulatory elements using word-counting or probabilistic methods (so-called “search by signal” methods) and the delineation of promoters by considering both sequence content and structural features (“search by content” methods). As an example of search by content, we explored in greater detail the association of promoters with CpG islands. However, due to differences in sequence content, the parameters used to detect CpG islands in humans and other vertebrates cannot be used for plants. Therefore, a preliminary attempt was made to define parameters that could possibly define CpG and CpNpG islands in Arabidopsis, by exploring the compositional landscape around the transcriptional start site. To this end, a data set of more than 5,000 gene sequences was built, including the promoter region, the 5′-untranslated region, and the first introns and coding exons. Preliminary analysis shows that promoter location based on the detection of potential CpG/CpNpG islands in the Arabidopsis genome is not straightforward. Nevertheless, because the landscape of CpG/CpNpG islands differs considerably between promoters and introns on the one side and exons (whether coding or not) on the other, more sophisticated approaches can probably be developed for the successful detection of “putative” CpG and CpNpG islands in plants. PMID:12857799

Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.

PubMed

Rentsendorj, Otgonchimeg; Nagy, Andrea; Sinkó, Ildikó; Daraba, Andreea; Barta, Endre; Kiss, Ibolya

2005-08-01

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.

Nucleotide sequence of the ribosomal RNA gene of Physarum polycephalum: intron 2 and its flanking regions of the 26S rRNA gene.

PubMed Central

Nomiyama, H; Kuhara, S; Kukita, T; Otsuka, T; Sakaki, Y

1981-01-01

The 26S ribosomal RNA gene of Physarum polycephalum is interrupted by two introns, and we have previously determined the sequence of one of them (intron 1) (Nomiyama et al. Proc.Natl.Acad.Sci.USA 78, 1376-1380, 1981). In this study we sequenced the second intron (intron 2) of about 0.5 kb length and its flanking regions, and found that one nucleotide at each junction is identical in intron 1 and intron 2, though the junction regions share no other sequence homology. Comparison of the flanking exon sequences to E. coli 23S rRNA sequences shows that conserved sequences are interspersed with tracts having little homology. In particular, the region encompassing the intron 2 interruption site is highly conserved. The E. coli ribosomal protein L1 binding region is also conserved. Images PMID:6171776

Exon–intron organization of genes in the slime mold Physarum polycephalum

PubMed Central

Trzcinska-Danielewicz, Joanna; Fronk, Jan

2000-01-01

The slime mold Physarum polycephalum is a morphologically simple organism with a large and complex genome. The exon–intron organization of its genes exhibits features typical for protists and fungi as well as those characteristic for the evolutionarily more advanced species. This indicates that both the taxonomic position as well as the size of the genome shape the exon–intron organization of an organism. The average gene has 3.7 introns which are on average 138 bp, with a rather narrow size distribution. Introns are enriched in AT base pairs by 13% relative to exons. The consensus sequences at exon–intron boundaries resemble those found for other species, with minor differences between short and long introns. A unique feature of P.polycephalum introns is the strong preference for pyrimidines in the coding strand throughout their length, without a particular enrichment at the 3′-ends. PMID:10982858

SURVEY AND SUMMARY: exon-intron organization of genes in the slime mold Physarum polycephalum.

PubMed

Trzcinska-Danielewicz, J; Fronk, J

2000-09-15

The slime mold Physarum polycephalum is a morphologically simple organism with a large and complex genome. The exon-intron organization of its genes exhibits features typical for protists and fungi as well as those characteristic for the evolutionarily more advanced species. This indicates that both the taxonomic position as well as the size of the genome shape the exon-intron organization of an organism. The average gene has 3.7 introns which are on average 138 bp, with a rather narrow size distribution. Introns are enriched in AT base pairs by 13% relative to exons. The consensus sequences at exon-intron boundaries resemble those found for other species, with minor differences between short and long introns. A unique feature of P.polycephalum introns is the strong preference for pyrimidines in the coding strand throughout their length, without a particular enrichment at the 3'-ends.

Evolution of Mhc-DRB introns: implications for the origin of primates.

PubMed

Kupfermann, H; Satta, Y; Takahata, N; Tichy, H; Klein, J

1999-06-01

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders-Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera.

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns.

PubMed

Gonzalez, P; Barroso, G; Labarère, J

1999-04-01

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.

RNA Sequencing of the Exercise Transcriptome in Equine Athletes

PubMed Central

Verini-Supplizi, Andrea; Barcaccia, Gianni; Albiero, Alessandro; D'Angelo, Michela; Campagna, Davide; Valle, Giorgio; Felicetti, Michela; Silvestrelli, Maurizio; Cappelli, Katia

2013-01-01

The horse is an optimal model organism for studying the genomic response to exercise-induced stress, due to its natural aptitude for athletic performance and the relative homogeneity of its genetic and environmental backgrounds. Here, we applied RNA-sequencing analysis through the use of SOLiD technology in an experimental framework centered on exercise-induced stress during endurance races in equine athletes. We monitored the transcriptional landscape by comparing gene expression levels between animals at rest and after competition. Overall, we observed a shift from coding to non-coding regions, suggesting that the stress response involves the differential expression of not annotated regions. Notably, we observed significant post-race increases of reads that correspond to repeats, especially the intergenic and intronic L1 and L2 transposable elements. We also observed increased expression of the antisense strands compared to the sense strands in intronic and regulatory regions (1 kb up- and downstream) of the genes, suggesting that antisense transcription could be one of the main mechanisms for transposon regulation in the horse under stress conditions. We identified a large number of transcripts corresponding to intergenic and intronic regions putatively associated with new transcriptional elements. Gene expression and pathway analysis allowed us to identify several biological processes and molecular functions that may be involved with exercise-induced stress. Ontology clustering reflected mechanisms that are already known to be stress activated (e.g., chemokine-type cytokines, Toll-like receptors, and kinases), as well as “nucleic acid binding” and “signal transduction activity” functions. There was also a general and transient decrease in the global rates of protein synthesis, which would be expected after strenuous global stress. In sum, our network analysis points toward the involvement of specific gene clusters in equine exercise-induced stress, including those involved in inflammation, cell signaling, and immune interactions. PMID:24391776

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

PubMed Central

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency. PMID:20042112

Forks in the tracks: Group II introns, spliceosomes, telomeres and beyond.

PubMed

Agrawal, Rajendra Kumar; Wang, Hong-Wei; Belfort, Marlene

2016-12-01

Group II introns are large catalytic RNAs that form a ribonucleoprotein (RNP) complex by binding to an intron-encoded protein (IEP). The IEP, which facilitates both RNA splicing and intron mobility, has multiple activities including reverse transcriptase. Recent structures of a group II intron RNP complex and of IEPs from diverse bacteria fuel arguments that group II introns are ancestrally related to eukaryotic spliceosomes as well as to telomerase and viruses. Furthermore, recent structural studies of various functional states of the spliceosome allow us to draw parallels between the group II intron RNP and the spliceosome. Here we present an overview of these studies, with an emphasis on the structure of the IEPs in their isolated and RNA-bound states and on their evolutionary relatedness. In addition, we address the conundrum of the free, albeit truncated IEPs forming dimers, whereas the IEP bound to the intron ribozyme is a monomer in the mature RNP. Future studies needed to resolve some of the outstanding issues related to group II intron RNP function and dynamics are also discussed.

Supervisory Control System Architecture for Advanced Small Modular Reactors

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

Cetiner, Sacit M; Cole, Daniel L; Fugate, David L

2013-08-01

This technical report was generated as a product of the Supervisory Control for Multi-Modular SMR Plants project within the Instrumentation, Control and Human-Machine Interface technology area under the Advanced Small Modular Reactor (SMR) Research and Development Program of the U.S. Department of Energy. The report documents the definition of strategies, functional elements, and the structural architecture of a supervisory control system for multi-modular advanced SMR (AdvSMR) plants. This research activity advances the state-of-the art by incorporating decision making into the supervisory control system architectural layers through the introduction of a tiered-plant system approach. The report provides a brief history ofmore » hierarchical functional architectures and the current state-of-the-art, describes a reference AdvSMR to show the dependencies between systems, presents a hierarchical structure for supervisory control, indicates the importance of understanding trip setpoints, applies a new theoretic approach for comparing architectures, identifies cyber security controls that should be addressed early in system design, and describes ongoing work to develop system requirements and hardware/software configurations.« less

A SNP uncoupling Mina expression from the TGFβ signaling pathway.

PubMed

Lian, Shang L; Mihi, Belgacem; Koyanagi, Madoka; Nakayama, Toshinori; Bix, Mark

2018-03-01

Mina is a JmjC family 2-oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell-type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1-region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1-region SNPs perturbs a Mina cis-regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus-spanning 26-kilobase genomic interval. We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c-but not C57Bl/6 allele-abolished both Smad3 binding and transforming growth factor beta (TGFβ) responsiveness. Our results demonstrate the TGFβ signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFβ signaling pathway. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

A SNP uncoupling Mina expression from the TGFβ signaling pathway

PubMed Central

Lian, Shang L.; Mihi, Belgacem; Koyanagi, Madoka; Nakayama, Toshinori

2017-01-01

Abstract Introduction Mina is a JmjC family 2‐oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell‐type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1‐region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. Methods Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1‐region SNPs perturbs a Mina cis‐regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus‐spanning 26‐kilobase genomic interval. Results We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c—but not C57Bl/6 allele—abolished both Smad3 binding and transforming growth factor beta (TGFβ) responsiveness. Conclusions Our results demonstrate the TGFβ signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFβ signaling pathway. PMID:28967702

The RNA helicase DDX17 controls the transcriptional activity of REST and the expression of proneural microRNAs in neuronal differentiation.

PubMed

Lambert, Marie-Pierre; Terrone, Sophie; Giraud, Guillaume; Benoit-Pilven, Clara; Cluet, David; Combaret, Valérie; Mortreux, Franck; Auboeuf, Didier; Bourgeois, Cyril F

2018-06-21

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.

Large Diameter Femoral Heads Impose Significant Alterations on the Strains Developed on Femoral Component and Bone: A Finite Element Analysis

PubMed Central

Theodorou, E.G; Provatidis, C.G; Babis, G.C; Georgiou, C.S; Megas, P.D

2011-01-01

Total Hip Arthroplasty aims at fully recreating a functional hip joint. Over the past years modular implant systems have become common practice and are widely used, due to the surgical options they provide. In addition Big Femoral Heads have also been implemented in the process, providing more flexibility for the surgeon. The current study aims at investigating the effects that femoral heads of bigger diameter may impose on the mechanical behavior of the bone-implant assembly. Using data acquired by Computed Tomographies and a Coordinate Measurement Machine, a cadaveric femur and a Profemur-E modular stem were fully digitized, leading to a three dimensional finite element model in ANSYS Workbench. Strains and stresses were then calculated, focusing on areas of clinical interest, based on Gruen zones: the calcar and the corresponding below the greater trochanter area in the proximal femur, the stem tip region and a profile line along linea aspera. The performed finite elements analysis revealed that the use of large diameter heads produces significant changes in strain development within the bone volume, especially in the lateral side. The application of Frost’s law in bone remodeling, validated the hypothesis that for all diameters normal bone growth occurs. However, in the calcar area lower strain values were recorded, when comparing with the reference model featuring a 28mm femoral head. Along line aspera and for the stem tip area, higher values were recorded. Finally, stresses calculated on the modular neck revealed increased values, but without reaching the yield strength of the titanium alloy used. PMID:21792381

Large diameter femoral heads impose significant alterations on the strains developed on femoral component and bone: a finite element analysis.

PubMed

Theodorou, E G; Provatidis, C G; Babis, G C; Georgiou, C S; Megas, P D

2011-01-01

Total Hip Arthroplasty aims at fully recreating a functional hip joint. Over the past years modular implant systems have become common practice and are widely used, due to the surgical options they provide. In addition Big Femoral Heads have also been implemented in the process, providing more flexibility for the surgeon. The current study aims at investigating the effects that femoral heads of bigger diameter may impose on the mechanical behavior of the bone-implant assembly. Using data acquired by Computed Tomographies and a Coordinate Measurement Machine, a cadaveric femur and a Profemur-E modular stem were fully digitized, leading to a three dimensional finite element model in ANSYS Workbench. Strains and stresses were then calculated, focusing on areas of clinical interest, based on Gruen zones: the calcar and the corresponding below the greater trochanter area in the proximal femur, the stem tip region and a profile line along linea aspera. The performed finite elements analysis revealed that the use of large diameter heads produces significant changes in strain development within the bone volume, especially in the lateral side. The application of Frost's law in bone remodeling, validated the hypothesis that for all diameters normal bone growth occurs. However, in the calcar area lower strain values were recorded, when comparing with the reference model featuring a 28mm femoral head. Along line aspera and for the stem tip area, higher values were recorded. Finally, stresses calculated on the modular neck revealed increased values, but without reaching the yield strength of the titanium alloy used.

Mitochondrial genes in the colourless alga Prototheca wickerhamii resemble plant genes in their exons but fungal genes in their introns.

PubMed Central

Wolff, G; Burger, G; Lang, B F; Kück, U

1993-01-01

The mitochondrial DNA from the colourless alga Prototheca wickerhamii contains two mosaic genes as was revealed from complete sequencing of the circular extranuclear genome. The genes for the large subunit of the ribosomal RNA (LSUrRNA) as well as for subunit I of the cytochrome oxidase (coxI) carry two and three intronic sequences respectively. On the basis of their canonical nucleotide sequences they can be classified as group I introns. Phylogenetic comparisons of the coxI protein sequences allow us to conclude that the P.wickerhamii mtDNA is much closer related to higher plant mtDNAs than to those of the chlorophyte alga C.reinhardtii. The comparison of the intron sequences revealed several unusual features: (1) The P.wickerhamii introns are structurally related to mitochondrial introns from various ascomycetous fungi. (2) Phylogenetic analyses indicate a close relationship between fungal and algal intronic sequences. (3) The P. wickerhamii introns are located at positions within the structural genes which can be considered as preferred intron insertion sites in homologous mitochondrial genes from fungi or liverwort. In all cases, the sequences adjacent to the insertion sites are very well conserved over large evolutionary distances. Our finding of highly similar introns in fungi and algae is consistent with the idea that introns have already been present in the bacterial ancestors of present day mitochondria and evolved concomitantly with the organelles. PMID:7680126

A modular finite-element model (MODFE) for areal and axisymmetric ground-water-flow problems, Part 2: Derivation of finite-element equations and comparisons with analytical solutions

USGS Publications Warehouse

Cooley, Richard L.

1992-01-01

MODFE, a modular finite-element model for simulating steady- or unsteady-state, area1 or axisymmetric flow of ground water in a heterogeneous anisotropic aquifer is documented in a three-part series of reports. In this report, part 2, the finite-element equations are derived by minimizing a functional of the difference between the true and approximate hydraulic head, which produces equations that are equivalent to those obtained by either classical variational or Galerkin techniques. Spatial finite elements are triangular with linear basis functions, and temporal finite elements are one dimensional with linear basis functions. Physical processes that can be represented by the model include (1) confined flow, unconfined flow (using the Dupuit approximation), or a combination of both; (2) leakage through either rigid or elastic confining units; (3) specified recharge or discharge at points, along lines, or areally; (4) flow across specified-flow, specified-head, or head-dependent boundaries; (5) decrease of aquifer thickness to zero under extreme water-table decline and increase of aquifer thickness from zero as the water table rises; and (6) head-dependent fluxes from springs, drainage wells, leakage across riverbeds or confining units combined with aquifer dewatering, and evapotranspiration. The matrix equations produced by the finite-element method are solved by the direct symmetric-Doolittle method or the iterative modified incomplete-Cholesky conjugate-gradient method. The direct method can be efficient for small- to medium-sized problems (less than about 500 nodes), and the iterative method is generally more efficient for larger-sized problems. Comparison of finite-element solutions with analytical solutions for five example problems demonstrates that the finite-element model can yield accurate solutions to ground-water flow problems.

A modular finite-element model (MODFE) for areal and axisymmetric ground-water-flow problems, Part 1: Model Description and User's Manual

USGS Publications Warehouse

Torak, L.J.

1993-01-01

A MODular, Finite-Element digital-computer program (MODFE) was developed to simulate steady or unsteady-state, two-dimensional or axisymmetric ground-water flow. Geometric- and hydrologic-aquifer characteristics in two spatial dimensions are represented by triangular finite elements and linear basis functions; one-dimensional finite elements and linear basis functions represent time. Finite-element matrix equations are solved by the direct symmetric-Doolittle method or the iterative modified, incomplete-Cholesky, conjugate-gradient method. Physical processes that can be represented by the model include (1) confined flow, unconfined flow (using the Dupuit approximation), or a combination of both; (2) leakage through either rigid or elastic confining beds; (3) specified recharge or discharge at points, along lines, and over areas; (4) flow across specified-flow, specified-head, or bead-dependent boundaries; (5) decrease of aquifer thickness to zero under extreme water-table decline and increase of aquifer thickness from zero as the water table rises; and (6) head-dependent fluxes from springs, drainage wells, leakage across riverbeds or confining beds combined with aquifer dewatering, and evapotranspiration. The report describes procedures for applying MODFE to ground-water-flow problems, simulation capabilities, and data preparation. Guidelines for designing the finite-element mesh and for node numbering and determining band widths are given. Tables are given that reference simulation capabilities to specific versions of MODFE. Examples of data input and model output for different versions of MODFE are provided.

A modular finite-element model (MODFE) for areal and axisymmetric ground-water-flow problems; Part 1, Model description and user's manual

USGS Publications Warehouse

Torak, Lynn J.

1992-01-01

A MODular, Finite-Element digital-computer program (MODFE) was developed to simulate steady or unsteady-state, two-dimensional or axisymmetric ground-water flow. Geometric- and hydrologic-aquifer characteristics in two spatial dimensions are represented by triangular finite elements and linear basis functions; one-dimensional finite elements and linear basis functions represent time. Finite-element matrix equations are solved by the direct symmetric-Doolittle method or the iterative modified, incomplete-Cholesky, conjugate-gradient method. Physical processes that can be represented by the model include (1) confined flow, unconfined flow (using the Dupuit approximation), or a combination of both; (2) leakage through either rigid or elastic confining beds; (3) specified recharge or discharge at points, along lines, and over areas; (4) flow across specified-flow, specified-head, or head-dependent boundaries; (5) decrease of aquifer thickness to zero under extreme water-table decline and increase of aquifer thickness from zero as the water table rises; and (6) head-dependent fluxes from springs, drainage wells, leakage across riverbeds or confining beds combined with aquifer dewatering, and evapotranspiration.The report describes procedures for applying MODFE to ground-water-flow problems, simulation capabilities, and data preparation. Guidelines for designing the finite-element mesh and for node numbering and determining band widths are given. Tables are given that reference simulation capabilities to specific versions of MODFE. Examples of data input and model output for different versions of MODFE are provided.

Parallel Loss of Plastid Introns and Their Maturase in the Genus Cuscuta

PubMed Central

McNeal, Joel R.; Kuehl, Jennifer V.; Boore, Jeffrey L.; Leebens-Mack, Jim; dePamphilis, Claude W.

2009-01-01

Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta. PMID:19543388

Parallel loss of plastid introns and their maturase in the genus Cuscuta.

PubMed

McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; Leebens-Mack, Jim; dePamphilis, Claude W

2009-06-19

Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.

Fungal origin by horizontal transfer of a plant mitochondrial group I intron in the chimeric CoxI gene of Peperomia.

PubMed

Vaughn, J C; Mason, M T; Sper-Whitis, G L; Kuhlman, P; Palmer, J D

1995-11-01

We present phylogenetic evidence that a group I intron in an angiosperm mitochondrial gene arose recently by horizontal transfer from a fungal donor species. A 1,716-bp fragment of the mitochondrial coxI gene from the angiosperm Peperomia polybotrya was amplified via the polymerase chain reaction and sequenced. Comparison to other coxI genes revealed a 966-bp group I intron, which, based on homology with the related yeast coxI intron aI4, potentially encodes a 279-amino-acid site-specific DNA endonuclease. This intron, which is believed to function as a ribozyme during its own splicing, is not present in any of 19 coxI genes examined from other diverse vascular plant species. Phylogenetic analysis of intron origin was carried out using three different tree-generating algorithms, and on a variety of nucleotide and amino acid data sets from the intron and its flanking exon sequences. These analyses show that the Peperomia coxI gene intron and exon sequences are of fundamentally different evolutionary origin. The Peperomia intron is more closely related to several fungal mitochondrial introns, two of which are located at identical positions in coxI, than to identically located coxI introns from the land plant Marchantia and the green alga Prototheca. Conversely, the exon sequence of this gene is, as expected, most closely related to other angiosperm coxI genes. These results, together with evidence suggestive of co-conversion of exonic markers immediately flanking the intron insertion site, lead us to conclude that the Peperomia coxI intron probably arose by horizontal transfer from a fungal donor, using the double-strand-break repair pathway. The donor species may have been one of the symbiotic mycorrhizal fungi that live in close obligate association with most plants.

[Detection of factor VIII intron 1 inversion in severe haemophilia A].

PubMed

Liang, Yan; Yan, Zhen-yu; Yan, Mei; Hua, Bao-lai; Xiao, Bai; Zhao, Yong-qiang; Liu, Jing-zhong

2009-06-01

Screening the intron 1 inversion of factor VIII (FVIII) in the population of severe haemophilia A(HA) in China and performing carrier detection and prenatal diagnosis. Using LD-PCR to detect intron 22 inversions and multiple-PCR within two tubes to intron 1 inversions in severe HA patients. Carrier detection and prenatal diagnosis were performed in affected families. Linkage analysis and DNA sequencing were used to verify these tests. One hundred and eighteen patients were seven diagnosed as intron 22 inversions and 7 were intron 1 inversions out of 247 severe HA patients. The prevalence of the intron 1 inversion in Chinese severe haemophilia A patients was 2.8% (7/247). Six women from family A and 2 from family B were diagnosed as carriers. One fetus from family A was affected fetus. Intron 1 inversion could be detected directly by multiple-PCR within two tubes. This method made the strategy more perfective in carrier and prenatal diagnosis of haemophilia A.

Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability.

PubMed

Bonnet, Amandine; Grosso, Ana R; Elkaoutari, Abdessamad; Coleno, Emeline; Presle, Adrien; Sridhara, Sreerama C; Janbon, Guilhem; Géli, Vincent; de Almeida, Sérgio F; Palancade, Benoit

2017-08-17

Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage. Copyright © 2017 Elsevier Inc. All rights reserved.

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron.

PubMed

Macreadie, I G; Scott, R M; Zinn, A R; Butow, R A

1985-06-01

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

PubMed

Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

2014-06-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Imprecise intron losses are less frequent than precise intron losses but are not rare in plants.

PubMed

Ma, Ming-Yue; Zhu, Tao; Li, Xue-Nan; Lan, Xin-Ran; Liu, Heng-Yuan; Yang, Yu-Fei; Niu, Deng-Ke

2015-05-27

In this study, we identified 19 intron losses, including 11 precise intron losses (PILs), six imprecise intron losses (IILs), one de-exonization, and one exon deletion in tomato and potato, and 17 IILs in Arabidopsis thaliana. Comparative analysis of related genomes confirmed that all of the IILs have been fixed during evolution. Consistent with previous studies, our results indicate that PILs are a major type of intron loss. However, at least in plants, IILs are unlikely to be as rare as previously reported. This article was reviewed by Jun Yu and Zhang Zhang. For complete reviews, see the Reviewers' Reports section.

Organellar maturases: A window into the evolution of the spliceosome.

PubMed

Schmitz-Linneweber, Christian; Lampe, Marie-Kristin; Sultan, Laure D; Ostersetzer-Biran, Oren

2015-09-01

During the evolution of eukaryotic genomes, many genes have been interrupted by intervening sequences (introns) that must be removed post-transcriptionally from RNA precursors to form mRNAs ready for translation. The origin of nuclear introns is still under debate, but one hypothesis is that the spliceosome and the intron-exon structure of genes have evolved from bacterial-type group II introns that invaded the eukaryotic genomes. The group II introns were most likely introduced into the eukaryotic genome from an α-proteobacterial predecessor of mitochondria early during the endosymbiosis event. These self-splicing and mobile introns spread through the eukaryotic genome and later degenerated. Pieces of introns became part of the general splicing machinery we know today as the spliceosome. In addition, group II introns likely brought intron maturases with them to the nucleus. Maturases are found in most bacterial introns, where they act as highly specific splicing factors for group II introns. In the spliceosome, the core protein Prp8 shows homology to group II intron-encoded maturases. While maturases are entirely intron specific, their descendant of the spliceosomal machinery, the Prp8 protein, is an extremely versatile splicing factor with multiple interacting proteins and RNAs. How could such a general player in spliceosomal splicing evolve from the monospecific bacterial maturases? Analysis of the organellar splicing machinery in plants may give clues on the evolution of nuclear splicing. Plants encode various proteins which are closely related to bacterial maturases. The organellar genomes contain one maturase each, named MatK in chloroplasts and MatR in mitochondria. In addition, several maturase genes have been found in the nucleus as well, which are acting on mitochondrial pre-RNAs. All plant maturases show sequence deviation from their progenitor bacterial maturases, and interestingly are all acting on multiple organellar group II intron targets. Moreover, they seem to function in the splicing of group II introns together with a number of additional nuclear-encoded splicing factors, possibly acting as an organellar proto-spliceosome. Together, this makes them interesting models for the early evolution of nuclear spliceosomal splicing. In this review, we summarize recent advances in our understanding of the role of plant maturases and their accessory factors in plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Insertion of a self-splicing intron into the mtDNA of atriploblastic animal

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

Valles, Y.; Halanych, K.; Boore, J.L.

2006-04-14

Nephtys longosetosa is a carnivorous polychaete worm that lives in the intertidal and subtidal zones with worldwide distribution (pleijel&rouse2001). Its mitochondrial genome has the characteristics typical of most metazoans: 37 genes; circular molecule; almost no intergenic sequence; and no significant gene rearrangements when compared to other annelid mtDNAs (booremoritz19981995). Ubiquitous features as small intergenic regions and lack of introns suggested that metazoan mtDNAs are under strong selective pressures to reduce their genome size allowing for faster replication requirements (booremoritz19981995Lynch2005). Yet, in 1996 two type I introns were found in the mtDNA of the basal metazoan Metridium senile (FigureX). Breaking amore » long-standing rule (absence of introns in metazoan mtDNA), this finding was later supported by the further presence of group I introns in other cnidarians. Interestingly, only the class Anthozoa within cnidarians seems to harbor such introns. Although several hundreds of triploblastic metazoan mtDNAs have been sequenced, this study is the first evidence of mitochondrial introns in triploblastic metazoans. The cox1 gene of N. longosetosa has an intron of almost 2 kbs in length. This finding represents as well the first instance of a group II intron (anthozoans harbor group I introns) in all metazoan lineages. Opposite trends are observed within plants, fungi and protist mtDNAs, where introns (both group I and II) and other non-coding sequences are widespread. Plant, fungal and protist mtDNA structure and organization differ enormously from that of metazoan mtDNA. Both, plant and fungal mtDNA are dynamic molecules that undergo high rates of recombination, contain long intergenic spacer regions and harbor both group I and group II introns. However, as metazoans they have a conserved gene content. Protists, on the other hand have a striking variation of gene content and introns that account for the genome size variation. In contrast to this mtDNA structure and organization diversity, current genome level studies point to a monophyletic origin of the mitochondria (REFS), raising questions such as: what are the pressures at work shaping the evolution of the mitochondrial genome at 'higher' levels? What drives the absence of introns and other non-coding spacers in metazoan mtDNA? What characteristics must have an intron to be maintained in an environment where 'extra chromosomes' are usually selected against?« less

Protein secondary structure prediction using modular reciprocal bidirectional recurrent neural networks.

PubMed

Babaei, Sepideh; Geranmayeh, Amir; Seyyedsalehi, Seyyed Ali

2010-12-01

The supervised learning of recurrent neural networks well-suited for prediction of protein secondary structures from the underlying amino acids sequence is studied. Modular reciprocal recurrent neural networks (MRR-NN) are proposed to model the strong correlations between adjacent secondary structure elements. Besides, a multilayer bidirectional recurrent neural network (MBR-NN) is introduced to capture the long-range intramolecular interactions between amino acids in formation of the secondary structure. The final modular prediction system is devised based on the interactive integration of the MRR-NN and the MBR-NN structures to arbitrarily engage the neighboring effects of the secondary structure types concurrent with memorizing the sequential dependencies of amino acids along the protein chain. The advanced combined network augments the percentage accuracy (Q₃) to 79.36% and boosts the segment overlap (SOV) up to 70.09% when tested on the PSIPRED dataset in three-fold cross-validation. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

A spatially localized architecture for fast and modular DNA computing

NASA Astrophysics Data System (ADS)

Chatterjee, Gourab; Dalchau, Neil; Muscat, Richard A.; Phillips, Andrew; Seelig, Georg

2017-09-01

Cells use spatial constraints to control and accelerate the flow of information in enzyme cascades and signalling networks. Synthetic silicon-based circuitry similarly relies on spatial constraints to process information. Here, we show that spatial organization can be a similarly powerful design principle for overcoming limitations of speed and modularity in engineered molecular circuits. We create logic gates and signal transmission lines by spatially arranging reactive DNA hairpins on a DNA origami. Signal propagation is demonstrated across transmission lines of different lengths and orientations and logic gates are modularly combined into circuits that establish the universality of our approach. Because reactions preferentially occur between neighbours, identical DNA hairpins can be reused across circuits. Co-localization of circuit elements decreases computation time from hours to minutes compared to circuits with diffusible components. Detailed computational models enable predictive circuit design. We anticipate our approach will motivate using spatial constraints for future molecular control circuit designs.

100-kA vacuum current breaker of a modular design

NASA Astrophysics Data System (ADS)

Ivanov, V. P.; Vozdvijenskii, V. A.; Jagnov, V. A.; Solodovnikov, S. G.; Mazulin, A. V.; Ryjkov, V. M.

1994-05-01

Direct current breaker of a modular design is developed for the strong field tokamak power supply system. The power supply system comprises four 800 MW alternative current generators with 4 GJ flywheels, thyristor rectifiers providing inductive stores pumping by a current up to 100 kA for 1 - 4 sec. To form current pulses of various shapes in the tokamak windings current breakers are used with either pneumatic or explosive drive, at a current switching synchronously of not worse than 100 mks. Current breakers of these types require that the current conducting elements be replaced after each shot. For recent years vacuum arc quenching chambers with an axial magnetic field are successfully employed as repetitive performance current breakers, basically for currents up to 40 kA. In the report some results of researches of a vacuum switch modular are presented which we used as prototype switch for currents of the order of 100 kA.

An interactive modular design for computerized photometry in spectrochemical analysis

NASA Technical Reports Server (NTRS)

Bair, V. L.

1980-01-01

A general functional description of totally automatic photometry of emission spectra is not available for an operating environment in which the sample compositions and analysis procedures are low-volume and non-routine. The advantages of using an interactive approach to computer control in such an operating environment are demonstrated. This approach includes modular subroutines selected at multiple-option, menu-style decision points. This style of programming is used to trace elemental determinations, including the automated reading of spectrographic plates produced by a 3.4 m Ebert mount spectrograph using a dc-arc in an argon atmosphere. The simplified control logic and modular subroutine approach facilitates innovative research and program development, yet is easily adapted to routine tasks. Operator confidence and control are increased by the built-in options including degree of automation, amount of intermediate data printed out, amount of user prompting, and multidirectional decision points.

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes.

PubMed

Valdes Franco, José A; Wang, Yi; Huo, Naxin; Ponciano, Grisel; Colvin, Howard A; McMahan, Colleen M; Gu, Yong Q; Belknap, William R

2018-04-19

Guayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome. The 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N 50  = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus. The sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

Mutually Exclusive Splicing of the Insect Dscam Pre-mRNA Directed by Competing Intronic RNA Secondary Structures

PubMed Central

Graveley, Brenton R.

2008-01-01

Summary Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. I have identified two classes of conserved elements in the Dscam exon 6 cluster, which contains 48 alternative exons—the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA. PMID:16213213

Estrogen receptor alpha regulates expression of the breast cancer 1 associated ring domain 1 (BARD1) gene through intronic DNA sequence.

PubMed

Creekmore, Amy L; Ziegler, Yvonne S; Bonéy, Jamie L; Nardulli, Ann M

2007-03-15

We have used a chromatin immunoprecipitation (ChIP)-based cloning strategy to isolate and identify genes associated with estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. One of the gene regions isolated was a 288bp fragment from the ninth intron of the breast cancer 1 associated ring domain (BARD1) gene. We demonstrated that ERalpha associated with this region of the endogenous BARD 1 gene in MCF-7 cells, that ERalpha bound to three of five ERE half sites located in the 288bp BARD1 region, and that this 288bp BARD1 region conferred estrogen responsiveness to a heterologous promoter. Importantly, treatment of MCF-7 cells with estrogen increased BARD1 mRNA and protein levels. These findings demonstrate that ChIP cloning strategies can be utilized to successfully isolate regulatory regions that are far removed from the transcription start site and assist in identifying cis elements involved in conferring estrogen responsiveness.

Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements

PubMed Central

Bridgewater, Laura C.; Walker, Marlan D.; Miller, Gwen C.; Ellison, Trevor A.; Holsinger, L. Daniel; Potter, Jennifer L.; Jackson, Todd L.; Chen, Reuben K.; Winkel, Vicki L.; Zhang, Zhaoping; McKinney, Sandra; de Crombrugghe, Benoit

2003-01-01

Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5′ region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled. PMID:12595563

Solar Pumped Solid State Lasers for Space Solar Power: Experimental Path

NASA Technical Reports Server (NTRS)

Fork, Richard L.; Carrington, Connie K.; Walker, Wesley W.; Cole, Spencer T.; Green, Jason J. A.; Laycock, Rustin L.

2003-01-01

We outline an experimentally based strategy designed to lead to solar pumped solid state laser oscillators useful for space solar power. Our method involves solar pumping a novel solid state gain element specifically designed to provide efficient conversion of sunlight in space to coherent laser light. Kilowatt and higher average power is sought from each gain element. Multiple such modular gain elements can be used to accumulate total average power of interest for power beaming in space, e.g., 100 kilowatts and more. Where desirable the high average power can also be produced as a train of pulses having high peak power (e.g., greater than 10(exp 10 watts). The modular nature of the basic gain element supports an experimental strategy in which the core technology can be validated by experiments on a single gain element. We propose to do this experimental validation both in terrestrial locations and also on a smaller scale in space. We describe a terrestrial experiment that includes diagnostics and the option of locating the laser beam path in vacuum environment. We describe a space based experiment designed to be compatible with the Japanese Experimental Module (JEM) on the International Space Station (ISS). We anticipate the gain elements will be based on low temperature (approx. 100 degrees Kelvin) operation of high thermal conductivity (k approx. 100 W/cm-K) diamond and sapphire (k approx. 4 W/cm-K). The basic gain element will be formed by sequences of thin alternating layers of diamond and Ti:sapphire with special attention given to the material interfaces. We anticipate this strategy will lead to a particularly simple, robust, and easily maintained low mass modelocked multi-element laser oscillator useful for space solar power.

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome.

PubMed

Schäfer, B; Merlos-Lange, A M; Anderl, C; Welser, F; Zimmer, M; Wolf, K

1991-01-01

In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain anar-14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.

An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

NASA Technical Reports Server (NTRS)

Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

1993-01-01

The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.

PubMed

Asakura, Yukari; Barkan, Alice

2007-12-01

The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.

Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

PubMed

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

PubMed Central

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns. PMID:24391881

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

PubMed

Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M

2013-07-01

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence in Podospora anserina†

PubMed Central

Begel, Odile; Boulay, Jocelyne; Albert, Beatrice; Dufour, Eric; Sainsard-Chanet, Annie

1999-01-01

Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (α) has been thought to play a prominent role in this syndrome. Intron α is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the α sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron α. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron α is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron α plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, “immortality” can be acquired not by the absence of intron α but rather by the lack of active cytochrome c oxidase. PMID:10330149

Molecular and bioinformatical characterization of a novel superfamily of cysteine-rich peptides from arthropods.

PubMed

Zeng, Xian-Chun; Nie, Yao; Luo, Xuesong; Wu, Shifen; Shi, Wanxia; Zhang, Lei; Liu, Yichen; Cao, Hanjun; Yang, Ye; Zhou, Jianping

2013-03-01

The full-length cDNA sequences of two novel cysteine-rich peptides (referred to as HsVx1 and MmKTx1) were obtained from scorpions. The two peptides represent a novel class of cysteine-rich peptides with a unique cysteine pattern. The genomic sequence of HsVx1 is composed of three exons interrupted by two introns that are localized in the mature peptide encoding region and inserted in phase 1 and phase 2, respectively. Such a genomic organization markedly differs from those of other peptides from scorpions described previously. Genome-wide search for the orthologs of HsVx1 identified 59 novel cysteine-rich peptides from arthropods. These peptides share a consistent cysteine pattern with HsVx1. Genomic comparison revealed extensive intron length differences and intronic number and position polymorphisms among the genes of these peptides. Further analysis identified 30 cases of intron sliding, 1 case of intron gain and 22 cases of intron loss occurred with the genes of the HsVx1 and HsVx1-like peptides. It is interesting to see that three HsVx1-like peptides XP_001658928, XP_001658929 and XP_001658930 were derived from a single gene (XP gene): the former two were generated from alternative splicing; the third one was encoded by a DNA region in the reverse complementary strand of the third intron of the XP gene. These findings strongly suggest that the genes of these cysteine-rich peptides were evolved by intron sliding, intron gain/loss, gene recombination and alternative splicing events in response to selective forces without changing their cysteine pattern. The evolution of these genes is dominated by intron sliding and intron loss. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular analysis of the split cox1 gene from the Basidiomycota Agrocybe aegerita: relationship of its introns with homologous Ascomycota introns and divergence levels from common ancestral copies.

PubMed

Gonzalez, P; Barroso, G; Labarère, J

1998-10-05

The Basidiomycota Agrocybe aegerita (Aa) mitochondrial cox1 gene (6790 nucleotides), encoding a protein of 527aa (58377Da), is split by four large subgroup IB introns possessing site-specific endonucleases assumed to be involved in intron mobility. When compared to other fungal COX1 proteins, the Aa protein is closely related to the COX1 one of the Basidiomycota Schizophyllum commune (Sc). This clade reveals a relationship with the studied Ascomycota ones, with the exception of Schizosaccharomyces pombe (Sp) which ranges in an out-group position compared with both higher fungi divisions. When comparison is extended to other kingdoms, fungal COX1 sequences are found to be more related to algae and plant ones (more than 57.5% aa similarity) than to animal sequences (53.6% aa similarity), contrasting with the previously established close relationship between fungi and animals, based on comparisons of nuclear genes. The four Aa cox1 introns are homologous to Ascomycota or algae cox1 introns sharing the same location within the exonic sequences. The percentages of identity of the intronic nucleotide sequences suggest a possible acquisition by lateral transfers of ancestral copies or of their derived sequences. These identities extend over the whole intronic sequences, arguing in favor of a transfer of the complete intron rather than a transfer limited to the encoded ORF. The intron i4 shares 74% of identity, at the nucleotidic level, with the Podospora anserina (Pa) intron i14, and up to 90.5% of aa similarity between the encoded proteins, i.e. the highest values reported to date between introns of two phylogenetically distant species. This low divergence argues for a recent lateral transfer between the two species. On the contrary, the low sequence identities (below 36%) observed between Aa i1 and the homologous Sp i1 or Prototheca wickeramii (Pw) i1 suggest a long evolution time after the separation of these sequences. The introns i2 and i3 possessed intermediate percentages of identity with their homologous Ascomycota introns. This is the first report of the complete nucleotide sequence and molecular organization of a mitochondrial cox1 gene of any member of the Basidiomycota division.

Intron Definition Is Required for Excision of the Minute Virus of Mice Small Intron and Definition of the Upstream Exon

PubMed Central

Haut, Donald D.; Pintel, D. J.

1998-01-01

Alternative splicing of pre-mRNAs plays a critical role in maximizing the coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing—using two donors (D1 and D2) and two acceptors (A1 and A2) within a region of 120 nucleotides—that determines the steady-state ratios of the various viral mRNAs. In this report, we show that the determinants that govern excision of the small intron are complex and are also required for efficient definition of the upstream exon. For the MVM small intron in its natural context, the two donors appear to compete for the splicing machinery: the position of D1 favors its usage, while the primary sequence of D2 must be more like the consensus sequence than is D1 to be used efficiently. We have genetically defined the branch points that are used for generation of the major and minor spliced forms and show that recognition of components of the small-intron acceptors is likely to be the dominant determinant in alternative small-intron excision. We have also identified a G-rich intronic enhancer sequence within the small intron that is essential for splicing of the minor form (D2 to A2) but not the major form (D1 to A1) of MVM mRNAs and is required for efficient definition of the upstream NS2-specific exon. In its natural context, the small intron appears to be excised by a mechanism consistent with intron definition. When the MVM small intron is expanded, various parameters of its excision are altered, indicating that critical cis-acting signals are context dependent. Relative use of the donors and acceptors is altered, and the upstream NS2-specific exon is no longer efficiently defined. The fact that definition of the upstream NS2-specific exon can be achieved by the MVM small intron in its natural context, but not when it is expanded, suggests that the multiple determinants that govern definition and excision of the small intron are required, in concert, for upstream exon definition. Our data are consistent with a model in which alternative splicing of the MVM P4-generated pre-mRNAs is governed by a hybrid of intron- and exon-defining mechanisms. PMID:9499034

Modular, Reconfigurable, and Rapid Response Space Systems: The Remote Sensing Advanced Technology Microsatellite

NASA Technical Reports Server (NTRS)

Esper, Jaime; Andary, Jim; Oberright, John; So, Maria; Wegner, Peter; Hauser, Joe

2004-01-01

Modular, Reconfigurable, and Rapid-response (MR(sup 2)) space systems represent a paradigm shift in the way space assets of all sizes are designed, manufactured, integrated, tested, and flown. This paper will describe the MR(sup 2) paradigm in detail, and will include guidelines for its implementation. The Remote Sensing Advanced Technology microsatellite (RSAT) is a proposed flight system test-bed used for developing and implementing principles and best practices for MR(sup 2) spacecraft, and their supporting infrastructure. The initial goal of this test-bed application is to produce a lightweight (approx. 100 kg), production-minded, cost-effective, and scalable remote sensing micro-satellite capable of high performance and broad applicability. Such applications range from future distributed space systems, to sensor-webs, and rapid-response satellite systems. Architectures will be explored that strike a balance between modularity and integration while preserving the MR(sup 2) paradigm. Modularity versus integration has always been a point of contention when approaching a design: whereas one-of-a-kind missions may require close integration resulting in performance optimization, multiple and flexible application spacecraft benefit &om modularity, resulting in maximum flexibility. The process of building spacecraft rapidly (< 7 days), requires a concerted and methodical look at system integration and test processes and pitfalls. Although the concept of modularity is not new and was first developed in the 1970s by NASA's Goddard Space Flight Center (Multi-Mission Modular Spacecraft), it was never modernized and was eventually abandoned. Such concepts as the Rapid Spacecraft Development Office (RSDO) became the preferred method for acquiring satellites. Notwithstanding, over the past 30 years technology has advanced considerably, and the time is ripe to reconsider modularity in its own right, as enabler of R(sup 2), and as a key element of transformational systems. The MR2 architecture provides a competitive advantage over the old modular approach in its rapid response to market needs that are difficult to predict both from the perspectives of evolving technology, as well as mission and application requirements.

Modular System to Enable Extravehicular Activity

NASA Technical Reports Server (NTRS)

Sargusingh, Miriam J.

2011-01-01

The ability to perform extravehicular activity (EVA), both human and robotic, has been identified as a key component to space missions to support such operations as assembly and maintenance of space system (e.g. construction and maintenance of the International Space Station), and unscheduled activities to repair an element of the transportation and habitation systems that can only be accessed externally and via unpressurized areas. In order to make human transportation beyond lower earth orbit (BLEO) practical, efficiencies must be incorporated into the integrated transportation systems to reduce system mass and operational complexity. Affordability is also a key aspect to be considered in space system development; this could be achieved through commonality, modularity and component reuse. Another key aspect identified for the EVA system was the ability to produce flight worthy hardware quickly to support early missions and near Earth technology demonstrations. This paper details a conceptual architecture for a modular extravehicular activity system (MEVAS) that would meet these stated needs for EVA capability that is affordable, and that could be produced relatively quickly. Operational concepts were developed to elaborate on the defined needs and define the key capabilities, operational and design constraints, and general timelines. The operational concept lead to a high level design concept for a module that interfaces with various space transportation elements and contains the hardware and systems required to support human and telerobotic EVA; the module would not be self-propelled and would rely on an interfacing element for consumable resources. The conceptual architecture was then compared to EVA Systems used in the Shuttle Orbiter, on the International Space Station to develop high level design concepts that incorporate opportunities for cost savings through hardware reuse, and quick production through the use of existing technologies and hardware designs. An upgrade option was included to make use of the developing suitport technologies.

Origin and evolution of SINEs in eukaryotic genomes.

PubMed

Kramerov, D A; Vassetzky, N S

2011-12-01

Short interspersed elements (SINEs) are one of the two most prolific mobile genomic elements in most of the higher eukaryotes. Although their biology is still not thoroughly understood, unusual life cycle of these simple elements amplified as genomic parasites makes their evolution unique in many ways. In contrast to most genetic elements including other transposons, SINEs emerged de novo many times in evolution from available molecules (for example, tRNA). The involvement of reverse transcription in their amplification cycle, huge number of genomic copies and modular structure allow variation mechanisms in SINEs uncommon or rare in other genetic elements (module exchange between SINE families, dimerization, and so on.). Overall, SINE evolution includes their emergence, progressive optimization and counteraction to the cell's defense against mobile genetic elements.

A multipurpose modular drone with adjustable arms produced via the FDM additive manufacturing process

NASA Astrophysics Data System (ADS)

Brischetto, Salvatore; Ciano, Alessandro; Ferro, Carlo Giovanni

2016-07-01

The present paper shows an innovative multirotor Unmanned Aerial Vehicle (UAV) which is able to easily and quickly change its configuration. In order to satisfy this feature, the principal structure is made of an universal plate, combined with a circular ring, to create a rail guide able to host the arms, in a variable number from 3 to 8, and the legs. The arms are adjustable and contain all the avionic and motor drivers to connect the main structure with each electric motor. The unique arm design, defined as all-in-one, allows classical single rotor configurations, double rotor configurations and amphibious configurations including inflatable elements positioned at the bottom of the arms. The proposed multi-rotor system is inexpensive because of the few universal pieces needed to compose the platform which allows the creation of a kit. This modular kit allows to have a modular drone with different configurations. Such configurations are distinguished among them for the number of arms, number of legs, number of rotors and motors, and landing capability. Another innovation feature is the introduction of the 3D printing technology to produce all the structural elements. In this manner, all the pieces are designed to be produced via the Fused Deposition Modelling (FDM) technology using desktop 3D printers. Therefore, an universal, dynamic and economic multi-rotor UAV has been developed.

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

PubMed

van Dolleweerd, Craig J; Kessans, Sarah A; Van de Bittner, Kyle C; Bustamante, Leyla Y; Bundela, Rudranuj; Scott, Barry; Nicholson, Matthew J; Parker, Emily J

2018-04-20

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

Modular microfluidic systems using reversibly attached PDMS fluid control modules

NASA Astrophysics Data System (ADS)

Skafte-Pedersen, Peder; Sip, Christopher G.; Folch, Albert; Dufva, Martin

2013-05-01

The use of soft lithography-based poly(dimethylsiloxane) (PDMS) valve systems is the dominating approach for high-density microscale fluidic control. Integrated systems enable complex flow control and large-scale integration, but lack modularity. In contrast, modular systems are attractive alternatives to integration because they can be tailored for different applications piecewise and without redesigning every element of the system. We present a method for reversibly coupling hard materials to soft lithography defined systems through self-aligning O-ring features thereby enabling easy interfacing of complex-valve-based systems with simpler detachable units. Using this scheme, we demonstrate the seamless interfacing of a PDMS-based fluid control module with hard polymer chips. In our system, 32 self-aligning O-ring features protruding from the PDMS fluid control module form chip-to-control module interconnections which are sealed by tightening four screws. The interconnection method is robust and supports complex fluidic operations in the reversibly attached passive chip. In addition, we developed a double-sided molding method for fabricating PDMS devices with integrated through-holes. The versatile system facilitates a wide range of applications due to the modular approach, where application specific passive chips can be readily attached to the flow control module.

Rare Autosomal Recessive Cardiac Valvular Form of Ehlers-Danlos Syndrome Results from Mutations in the COL1A2 Gene That Activate the Nonsense-Mediated RNA Decay Pathway

PubMed Central

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A.; Shinkai, Hiroshi; Hoyme, H. Eugene; Pyeritz, Reed E.; Byers, Peter H.

2004-01-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of proα2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement. PMID:15077201

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.

PubMed

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A; Shinkai, Hiroshi; Hoyme, H Eugene; Pyeritz, Reed E; Byers, Peter H

2004-05-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

Fragile X mental retardation 1 (FMR1) intron 1 methylation in blood predicts verbal cognitive impairment in female carriers of expanded FMR1 alleles: evidence from a pilot study.

PubMed

Godler, David E; Slater, Howard R; Bui, Quang M; Storey, Elsdon; Ono, Michele Y; Gehling, Freya; Inaba, Yoshimi; Francis, David; Hopper, John L; Kinsella, Glynda; Amor, David J; Hagerman, Randi J; Loesch, Danuta Z

2012-03-01

Cognitive status in females with mutations in the FMR1 (fragile X mental retardation 1) gene is highly variable. A biomarker would be of value for predicting which individuals were liable to develop cognitive impairment and could benefit from early intervention. A detailed analysis of CpG sites bridging exon 1 and intron 1 of FMR1, known as fragile X-related epigenetic element 2 (FREE2), suggests that a simple blood test could identify these individuals. Study participants included 74 control females (<40 CGG repeats), 62 premutation (PM) females (55-200 CGG repeats), and 18 full-mutation (FM) females assessed with Wechsler intelligence quotient (IQ) tests. We used MALDI-TOF mass spectrometry to determine the methylation status of FREE2 CpG sites that best identified low-functioning (IQ <70) FM females (>200 CGG repeats), compared the results with those for Southern blot FMR1 activation ratios, and related these assessments to the level of production of the FMR1 protein product in blood. A methylation analysis of intron 1 CpG sites 10-12 showed the highest diagnostic sensitivity (100%) and specificity (98%) of all the molecular measures tested for detecting females with a standardized verbal IQ of <70 among the study participants. In the group consisting of only FM females, methylation of these sites was significantly correlated with full-scale IQ, verbal IQ, and performance IQ. Several verbal subtest scores showed strong correlation with the methylation of these sites (P = 1.2 × 10(-5)) after adjustment for multiple measures. The data suggest that hypermethylation of the FMR1 intron 1 sites in blood is predictive of cognitive impairment in FM females, with implications for improved fragile X syndrome diagnostics in young children and screening of the newborn population.

Activation of protein kinase C induces nuclear translocation of RFX1 and down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60 cells.

PubMed

Chen, L; Smith, L; Johnson, M R; Wang, K; Diasio, R B; Smith, J B

2000-10-13

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inhibition of Myc synthesis accounted for the drop in Myc protein, because PMA treatment had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein complexes. Increased MIE1 binding was independent of protein synthesis and abolished by a selective PKC inhibitor, which also prevented the effect of PMA on myc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol without affecting total RFX1. Transfection of HL-60 cells with myc reporter gene constructs showed that the RFX1-binding X box was required for the down-regulation of reporter gene expression by PMA. These findings suggest that nuclear translocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 cells.

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

PubMed

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

Factors influencing alternative splice site utilization in vivo.

PubMed Central

Fu, X Y; Manley, J L

1987-01-01

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA. Images PMID:3029566

Structural analysis of the 5{prime} region of mouse and human Huntington disease genes reveals conservation of putative promoter region and Di- and trinucleotide polymorphisms

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

Lin, Biaoyang; Nasir, J.; Kalchman, M.A.

1995-02-10

We have previously cloned and characterized the murine homologue of the Huntington disease (HD) gene and shown that it maps to mouse chromosome 5 within a region of conserved synteny with human chromosome 4p16.3. Here we present a detailed comparison of the sequence of the putative promoter and the organization of the 5{prime} genomic region of the murine (Hdh) and human HD genes encompassing the first five exons. We show that in this region these two genes share identical exon boundaries, but have different-size introns. Two dinucleotide (CT) and one trinucleotide intronic polymorphism in Hdh and an intronic CA polymorphismmore » in the HD gene were identified. Comparison of 940-bp sequence 5{prime} to the putative translation start site reveals a highly conserved region (78.8% nucleotide identity) between Hdh and the HD gene from nucleotide -56 to -206 (of Hdh). Neither Hdh nor the HD gene have typical TATA or CCAAT elements, but both show one putative AP2 binding site and numerous potential Sp1 binding sites. The high sequence identity between Hdh and the HD gene for approximately 200 bp 5{prime} to the putative translation start site indicates that these sequences may play a role in regulating expression of the Huntington disease gene. 30 refs., 4 figs., 2 tabs.« less

An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds.

PubMed

Haase, B; Jude, R; Brooks, S A; Leeb, T

2008-06-01

The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele (To) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of horses with the tobiano pattern did not show the polymorphism in intron 13 and consequently the test was not useful to identify putative homozygotes for To within this breed. Speculations were raised that an independent mutation might cause the tobiano spotting pattern in this breed. Recently, the putative causative mutation for To was described as a large chromosomal inversion on equine chromosome 3. One of the inversion breakpoints is approximately 70 kb downstream of the KIT gene and probably disrupts a regulatory element of the KIT gene. We obtained genotypes for the intron 13 SNP and the chromosomal inversion for 204 tobiano spotted horses and 24 control animals of several breeds. The genotyping data confirmed that the chromosomal inversion was perfectly associated with the To allele in all investigated horses. Therefore, the new test is suitable to discriminate heterozygous To/+ and homozygous To/To horses in the investigated breeds.

Comprehensive profiling of rhizome-associated alternative splicing and alternative polyadenylation in moso bamboo (Phyllostachys edulis).

PubMed

Wang, Taotao; Wang, Huiyuan; Cai, Dawei; Gao, Yubang; Zhang, Hangxiao; Wang, Yongsheng; Lin, Chentao; Ma, Liuyin; Gu, Lianfeng

2017-08-01

Moso bamboo (Phyllostachys edulis) represents one of the fastest-spreading plants in the world, due in part to its well-developed rhizome system. However, the post-transcriptional mechanism for the development of the rhizome system in bamboo has not been comprehensively studied. We therefore used a combination of single-molecule long-read sequencing technology and polyadenylation site sequencing (PAS-seq) to re-annotate the bamboo genome, and identify genome-wide alternative splicing (AS) and alternative polyadenylation (APA) in the rhizome system. In total, 145 522 mapped full-length non-chimeric (FLNC) reads were analyzed, resulting in the correction of 2241 mis-annotated genes and the identification of 8091 previously unannotated loci. Notably, more than 42 280 distinct splicing isoforms were derived from 128 667 intron-containing full-length FLNC reads, including a large number of AS events associated with rhizome systems. In addition, we characterized 25 069 polyadenylation sites from 11 450 genes, 6311 of which have APA sites. Further analysis of intronic polyadenylation revealed that LTR/Gypsy and LTR/Copia were two major transposable elements within the intronic polyadenylation region. Furthermore, this study provided a quantitative atlas of poly(A) usage. Several hundred differential poly(A) sites in the rhizome-root system were identified. Taken together, these results suggest that post-transcriptional regulation may potentially have a vital role in the underground rhizome-root system. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Multiple splicing defects in an intronic false exon.

PubMed

Sun, H; Chasin, L A

2000-09-01

Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

PubMed

Liu, Xiaochuan; Freitas, Jaime; Zheng, Dinghai; Oliveira, Marta S; Hoque, Mainul; Martins, Torcato; Henriques, Telmo; Tian, Bin; Moreira, Alexandra

2017-12-01

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster , expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis -element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215 C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate. © 2017 Liu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

PubMed Central

Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

2012-01-01

Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3′ and 5′ splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

Enantioselective total synthesis of hyperforin.

PubMed

Sparling, Brian A; Moebius, David C; Shair, Matthew D

2013-01-16

A modular, 18-step total synthesis of hyperforin is described. The natural product was quickly accessed using latent symmetry elements, whereby a group-selective, Lewis acid-catalyzed epoxide-opening cascade cyclization was used to furnish the bicyclo[3.3.1]nonane core and set two key quaternary stereocenters.

Modular space station phase B extension preliminary system design. Volume 2: Operations and crew analyses

NASA Technical Reports Server (NTRS)

Meston, R. D.; Schall, M. R., Jr.; Brockman, C. L.; Bender, W. H.

1972-01-01

All analyses and tradeoffs conducted to establish the MSS operations and crew activities are discussed. The missions and subsystem integrated analyses that were completed to assure compatibility of program elements and consistency with program objectives are presented.

Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.

PubMed

Asakura, Yukari; Bayraktar, Omer Ali; Barkan, Alice

2008-11-01

Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.

Polymorphism in Mitochondrial Group I Introns among Cryptococcus neoformans and Cryptococcus gattii Genotypes and Its Association with Drug Susceptibility.

PubMed

Gomes, Felipe E E S; Arantes, Thales D; Fernandes, José A L; Ferreira, Leonardo C; Romero, Héctor; Bosco, Sandra M G; Oliveira, Maria T B; Del Negro, Gilda M B; Theodoro, Raquel C

2018-01-01

Cryptococcosis, one of the most important systemic mycosis in the world, is caused by different genotypes of Cryptococcus neoformans and Cryptococcus gattii , which differ in their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i) they are polymorphic sequences; (ii) their self-splicing is inhibited by some drugs; and (iii) their correct splicing under parasitic conditions is indispensable for pathogen survival. Here, we investigated the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus isolates and its possible relation to drug susceptibility. Sequencing revealed two new introns in the LSU rRNA gene. All the introns showed high sequence similarity to other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient non-allelic invasion. Intron presence was statistically associated with those genotypes reported to be less pathogenic ( p < 0.001). Further virulence assays are needed to confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU rRNA introns may influence the minimum inhibitory concentration (MIC) of amphotericin B and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome of Cryptococcus as potential molecular markers for antifungal resistance, as well as therapeutic targets.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria

PubMed Central

Sultan, Laure D.; Grewe, Felix; Rolle, Katarzyna; Abudraham, Sivan; Shevtsov, Sofia; Klipcan, Liron; Barciszewski, Jan; Dietrich, André

2016-01-01

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory. PMID:27760804

Alternative splicing by participation of the group II intron ORF in extremely halotolerant and alkaliphilic Oceanobacillus iheyensis.

PubMed

Chee, Gab-Joo; Takami, Hideto

2011-01-01

Group II introns inserted into genes often undergo splicing at unexpected sites, and participate in the transcription of host genes. We identified five copies of a group II intron, designated Oi.Int, in the genome of an extremely halotolerant and alkaliphilic bacillus, Oceanobacillus iheyensis. The Oi.Int4 differs from the Oi.Int3 at four bases. The ligated exons of the Oi.Int4 could not be detected by RT-PCR assays in vivo or in vitro although group II introns can generally self-splice in vitro without the involvement of an intron-encoded open reading frame (ORF). In the Oi.Int4 mutants with base substitutions within the ORF, ligated exons were detected by in vitro self-splicing. It was clear that the ligation of exons during splicing is affected by the sequence of the intron-encoded ORF since the splice sites corresponded to the joining sites of the intron. In addition, the mutant introns showed unexpected multiple products with alternative 5' splice sites. These findings imply that alternative 5' splicing which causes a functional change of ligated exons presumably has influenced past adaptations of O. iheyensis to various environmental changes.

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer

PubMed Central

Nisa-Martínez, Rafael; Jiménez-Zurdo, José I.; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás

2007-01-01

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase–maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (ΔORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic ΔORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature. PMID:17158161

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer.

PubMed

Nisa-Martínez, Rafael; Jiménez-Zurdo, José I; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás

2007-01-01

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture.

PubMed

Pai, Athma A; Henriques, Telmo; McCue, Kayla; Burkholder, Adam; Adelman, Karen; Burge, Christopher B

2017-12-27

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning ('intron definition') or exon-spanning ('exon definition') pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila , using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60-70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly low variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.

Determinism and randomness in the evolution of introns and sine inserts in mouse and human mitochondrial solute carrier and cytokine receptor genes.

PubMed

Cianciulli, Antonia; Calvello, Rosa; Panaro, Maria A

2015-04-01

In the homologous genes studied, the exons and introns alternated in the same order in mouse and human. We studied, in both species: corresponding short segments of introns, whole corresponding introns and complete homologous genes. We considered the total number of nucleotides and the number and orientation of the SINE inserts. Comparisons of mouse and human data series showed that at the level of individual relatively short segments of intronic sequences the stochastic variability prevails in the local structuring, but at higher levels of organization a deterministic component emerges, conserved in mouse and human during the divergent evolution, despite the ample re-editing of the intronic sequences and the fact that processes such as SINE spread had taken place in an independent way in the two species. Intron conservation is negatively correlated with the SINE occupancy, suggesting that virus inserts interfere with the conservation of the sequences inherited from the common ancestor. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evolution of the myosin heavy chain gene MYH14 and its intronic microRNA miR-499: muscle-specific miR-499 expression persists in the absence of the ancestral host gene.

PubMed

Bhuiyan, Sharmin Siddique; Kinoshita, Shigeharu; Wongwarangkana, Chaninya; Asaduzzaman, Md; Asakawa, Shuichi; Watabe, Shugo

2013-07-06

A novel sarcomeric myosin heavy chain gene, MYH14, was identified following the completion of the human genome project. MYH14 contains an intronic microRNA, miR-499, which is expressed in a slow/cardiac muscle specific manner along with its host gene; it plays a key role in muscle fiber-type specification in mammals. Interestingly, teleost fish genomes contain multiple MYH14 and miR-499 paralogs. However, the evolutionary history of MYH14 and miR-499 has not been studied in detail. In the present study, we identified MYH14/miR-499 loci on various teleost fish genomes and examined their evolutionary history by sequence and expression analyses. Synteny and phylogenetic analyses depict the evolutionary history of MYH14/miR-499 loci where teleost specific duplication and several subsequent rounds of species-specific gene loss events took place. Interestingly, miR-499 was not located in the MYH14 introns of certain teleost fish. An MYH14 paralog, lacking miR-499, exhibited an accelerated rate of evolution compared with those containing miR-499, suggesting a putative functional relationship between MYH14 and miR-499. In medaka, Oryzias latipes, miR-499 is present where MYH14 is completely absent in the genome. Furthermore, by using in situ hybridization and small RNA sequencing, miR-499 was expressed in the notochord at the medaka embryonic stage and slow/cardiac muscle at the larval and adult stages. Comparing the flanking sequences of MYH14/miR-499 loci between torafugu Takifugu rubripes, zebrafish Danio rerio, and medaka revealed some highly conserved regions, suggesting that cis-regulatory elements have been functionally conserved in medaka miR-499 despite the loss of its host gene. This study reveals the evolutionary history of the MYH14/miRNA-499 locus in teleost fish, indicating divergent distribution and expression of MYH14 and miR-499 genes in different teleost fish lineages. We also found that medaka miR-499 was even expressed in the absence of its host gene. To our knowledge, this is the first report that shows the conversion of intronic into non-intronic miRNA during the evolution of a teleost fish lineage.

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

PubMed

Melangath, Geetha; Sen, Titash; Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha; Vijayraghavan, Usha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing

PubMed Central

Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3’ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5’ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5’ss in dtd1+ intron 1 and of an upstream alternative 3’ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5’ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5’ ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3’ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast. PMID:29236736

Evolution of RNA-Protein Interactions: Non-Specific Binding Led to RNA Splicing Activity of Fungal Mitochondrial Tyrosyl-tRNA Synthetases

PubMed Central

Lamech, Lilian T.; Mallam, Anna L.; Lambowitz, Alan M.

2014-01-01

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was “fixed” by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins. PMID:25536042

Evolution of RNA-protein interactions: non-specific binding led to RNA splicing activity of fungal mitochondrial tyrosyl-tRNA synthetases.

PubMed

Lamech, Lilian T; Mallam, Anna L; Lambowitz, Alan M

2014-12-01

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was "fixed" by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.

Isolation and identification of gene-specific microRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2006-01-01

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof- of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

Isolation and identification of gene-specific microRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2013-01-01

Computer programming has identified hundreds of genomic hairpin sequences, many with functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA generation system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafishes, chicken embryos, and adult mice. We have also developed an miRNA isolation protocol, based on the complementarity between the designed miRNA and its target gene sequence, to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proven-of-principle method, we now have full knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing systems.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium.

PubMed

Torrado, Mario; Iglesias, Raquel; Nespereira, Beatriz; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T

2009-07-01

The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers.

PubMed

Dutta, Debargh; Gunasekera, Devi; Ragni, Margaret V; Pratt, Kathleen P

2016-12-27

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ∼50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers. The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8 sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from F8 RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from F8 exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing F8 exon 1 and the VBP1 gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based "inverse-shifting" PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect F8 inversion mutations via same-day testing of patient samples.

Block-Module Electric Machines of Alternating Current

NASA Astrophysics Data System (ADS)

Zabora, I.

2018-03-01

The paper deals with electric machines having active zone based on uniform elements. It presents data on disk-type asynchronous electric motors with short-circuited rotors, where active elements are made by integrated technique that forms modular elements. Photolithography, spraying, stamping of windings, pressing of core and combined methods are utilized as the basic technological approaches of production. The constructions and features of operation for new electric machine - compatible electric machines-transformers are considered. Induction motors are intended for operation in hermetic plants with extreme conditions surrounding gas, steam-to-gas and liquid environment at a high temperature (to several hundred of degrees).

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

PubMed

Sultan, Laure D; Mileshina, Daria; Grewe, Felix; Rolle, Katarzyna; Abudraham, Sivan; Głodowicz, Paweł; Niazi, Adnan Khan; Keren, Ido; Shevtsov, Sofia; Klipcan, Liron; Barciszewski, Jan; Mower, Jeffrey P; Dietrich, André; Ostersetzer-Biran, Oren

2016-11-01

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory. © 2016 American Society of Plant Biologists. All rights reserved.

Mollusk genes encoding lysine tRNA (UUU) contain introns.

PubMed

Matsuo, M; Abe, Y; Saruta, Y; Okada, N

1995-11-20

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Processing of Archaebacterial Intron-Containing tRNA Gene Transcripts.

DTIC Science & Technology

1987-07-31

1{ 1. Project Goals: A. To determine the mechanism of tRNA intron processing in the halophilic archaebacteria. B. Characterize and compare the...enzyme(s) responsible for the removal of 5’-flanking sequences from halophilic and sulfur-dependent tRNA gene transcripts. C. Examine the structure and...distribution of tRNA introns in the halophilic archaebacteria. 2. Accomplishments: A. Intron processing mechanism We have succeeded in our primary

Pre-Mrna Introns as a Model for Cryptographic Algorithm:. Theory and Experiments

NASA Astrophysics Data System (ADS)

Regoli, Massimo

2010-01-01

The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. In particular the RNA sequences have some sections called Introns. Introns, derived from the term "intragenic regions", are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by Biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behaviour in the access to the secret key to code the messages. In the RNA-Crypto System algorithm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

a Simple Symmetric Algorithm Using a Likeness with Introns Behavior in RNA Sequences

NASA Astrophysics Data System (ADS)

Regoli, Massimo

2009-02-01

The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. The RNA sequences has some sections called Introns. Introns, derived from the term "intragenic regions", are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by Biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behaviour in the access to the secret key to code the messages. In the RNA-Crypto System algoritnm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles.

PubMed

Molina-Sánchez, Maria D; García-Rodríguez, Fernando M; Toro, Nicolás

2016-01-01

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3' end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro . The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles

PubMed Central

Molina-Sánchez, Maria D.; García-Rodríguez, Fernando M.; Toro, Nicolás

2016-01-01

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3′ end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods. PMID:27730127

Evolutionary and Developmental Modules

PubMed Central

Lacquaniti, Francesco; Ivanenko, Yuri P.; d’Avella, Andrea; Zelik, Karl E.; Zago, Myrka

2013-01-01

The identification of biological modules at the systems level often follows top-down decomposition of a task goal, or bottom-up decomposition of multidimensional data arrays into basic elements or patterns representing shared features. These approaches traditionally have been applied to mature, fully developed systems. Here we review some results from two other perspectives on modularity, namely the developmental and evolutionary perspective. There is growing evidence that modular units of development were highly preserved and recombined during evolution. We first consider a few examples of modules well identifiable from morphology. Next we consider the more difficult issue of identifying functional developmental modules. We dwell especially on modular control of locomotion to argue that the building blocks used to construct different locomotor behaviors are similar across several animal species, presumably related to ancestral neural networks of command. A recurrent theme from comparative studies is that the developmental addition of new premotor modules underlies the postnatal acquisition and refinement of several different motor behaviors in vertebrates. PMID:23730285

Evolutionary and developmental modules.

PubMed

Lacquaniti, Francesco; Ivanenko, Yuri P; d'Avella, Andrea; Zelik, Karl E; Zago, Myrka

2013-01-01

The identification of biological modules at the systems level often follows top-down decomposition of a task goal, or bottom-up decomposition of multidimensional data arrays into basic elements or patterns representing shared features. These approaches traditionally have been applied to mature, fully developed systems. Here we review some results from two other perspectives on modularity, namely the developmental and evolutionary perspective. There is growing evidence that modular units of development were highly preserved and recombined during evolution. We first consider a few examples of modules well identifiable from morphology. Next we consider the more difficult issue of identifying functional developmental modules. We dwell especially on modular control of locomotion to argue that the building blocks used to construct different locomotor behaviors are similar across several animal species, presumably related to ancestral neural networks of command. A recurrent theme from comparative studies is that the developmental addition of new premotor modules underlies the postnatal acquisition and refinement of several different motor behaviors in vertebrates.

Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology.

PubMed

Wang, Baojun; Kitney, Richard I; Joly, Nicolas; Buck, Martin

2011-10-18

Modular and orthogonal genetic logic gates are essential for building robust biologically based digital devices to customize cell signalling in synthetic biology. Here we constructed an orthogonal AND gate in Escherichia coli using a novel hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ(54)-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate. The circuits were assembled using a parts-based engineering approach of quantitative characterization, modelling, followed by construction and testing. The results show that new genetic logic devices can be engineered predictably from novel native orthogonal biological control elements using quantitatively in-context characterized parts. © 2011 Macmillan Publishers Limited. All rights reserved.

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.

Origin and evolution of SINEs in eukaryotic genomes

PubMed Central

Kramerov, D A; Vassetzky, N S

2011-01-01

Short interspersed elements (SINEs) are one of the two most prolific mobile genomic elements in most of the higher eukaryotes. Although their biology is still not thoroughly understood, unusual life cycle of these simple elements amplified as genomic parasites makes their evolution unique in many ways. In contrast to most genetic elements including other transposons, SINEs emerged de novo many times in evolution from available molecules (for example, tRNA). The involvement of reverse transcription in their amplification cycle, huge number of genomic copies and modular structure allow variation mechanisms in SINEs uncommon or rare in other genetic elements (module exchange between SINE families, dimerization, and so on.). Overall, SINE evolution includes their emergence, progressive optimization and counteraction to the cell's defense against mobile genetic elements. PMID:21673742

The in vivo use of alternate 3'-splice sites in group I introns.

PubMed

Sellem, C H; Belcour, L

1994-04-11

Alternative splicing of group I introns has been postulated as a possible mechanism that would ensure the translation of proteins encoded into intronic open reading frames, discontinuous with the upstream exon and lacking an initiation signal. Alternate splice sites were previously depicted according to secondary structures of several group I introns. We present here strong evidence that, in the case of Podospora anserina nad 1-i4 and cox1-i7 mitochondrial introns, alternative splicing events do occur in vivo. Indeed, by PCR experiments we have detected molecules whose sequence is precisely that expected if the predicted alternate 3'-splice sites were used.

FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster.

PubMed

Moschetti, R; Marsano, R M; Barsanti, P; Caggese, C; Caizzi, R

2004-05-01

Foldback ( FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w(67C23), a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w(67C23) locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.

The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture

PubMed Central

Pai, Athma A; Henriques, Telmo; McCue, Kayla; Burkholder, Adam; Adelman, Karen

2017-01-01

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning (‘intron definition’) or exon-spanning (‘exon definition’) pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60–70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly low variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing. PMID:29280736

The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture

DOE PAGES

Pai, Athma A.; Henriques, Telmo; McCue, Kayla; ...

2017-12-27

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning (‘intron definition’) or exon-spanning (‘exon definition’) pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60–70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly lowmore » variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.« less

The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture

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

Pai, Athma A.; Henriques, Telmo; McCue, Kayla

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning (‘intron definition’) or exon-spanning (‘exon definition’) pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60–70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly lowmore » variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.« less

RNA-SeQC: RNA-seq metrics for quality control and process optimization.

PubMed

DeLuca, David S; Levin, Joshua Z; Sivachenko, Andrey; Fennell, Timothy; Nazaire, Marc-Danie; Williams, Chris; Reich, Michael; Winckler, Wendy; Getz, Gad

2012-06-01

RNA-seq, the application of next-generation sequencing to RNA, provides transcriptome-wide characterization of cellular activity. Assessment of sequencing performance and library quality is critical to the interpretation of RNA-seq data, yet few tools exist to address this issue. We introduce RNA-SeQC, a program which provides key measures of data quality. These metrics include yield, alignment and duplication rates; GC bias, rRNA content, regions of alignment (exon, intron and intragenic), continuity of coverage, 3'/5' bias and count of detectable transcripts, among others. The software provides multi-sample evaluation of library construction protocols, input materials and other experimental parameters. The modularity of the software enables pipeline integration and the routine monitoring of key measures of data quality such as the number of alignable reads, duplication rates and rRNA contamination. RNA-SeQC allows investigators to make informed decisions about sample inclusion in downstream analysis. In summary, RNA-SeQC provides quality control measures critical to experiment design, process optimization and downstream computational analysis. See www.genepattern.org to run online, or www.broadinstitute.org/rna-seqc/ for a command line tool.

Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals

PubMed Central

Pridans, Clare; Lillico, Simon; Whitelaw, Bruce; Hume, David A

2014-01-01

The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE). We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large “cargo” for applications in gene therapy and vaccine delivery. PMID:26015955

Development of Intelligent Method for Differential Diagnosis of Specific Language Impairment

DTIC Science & Technology

2001-10-25

Grant Number Program Element Number Author(s) Project Number Task Number Work Unit Number Performing Organization Name(s) and Address(es) Dept...Development in a Grammatical SLI boy: Modularity and Innateness, Journal of Neurolinguistics 10, (1997) 75-107. [8] J.P. Craiger, D.F. Goodman, R.J

Short interspersed nuclear elements (SINEs) are abundant in Solanaceae and have a family-specific impact on gene structure and genome organization.

PubMed

Seibt, Kathrin M; Wenke, Torsten; Muders, Katja; Truberg, Bernd; Schmidt, Thomas

2016-05-01

Short interspersed nuclear elements (SINEs) are highly abundant non-autonomous retrotransposons that are widespread in plants. They are short in size, non-coding, show high sequence diversity, and are therefore mostly not or not correctly annotated in plant genome sequences. Hence, comparative studies on genomic SINE populations are rare. To explore the structural organization and impact of SINEs, we comparatively investigated the genome sequences of the Solanaceae species potato (Solanum tuberosum), tomato (Solanum lycopersicum), wild tomato (Solanum pennellii), and two pepper cultivars (Capsicum annuum). Based on 8.5 Gbp sequence data, we annotated 82 983 SINE copies belonging to 10 families and subfamilies on a base pair level. Solanaceae SINEs are dispersed over all chromosomes with enrichments in distal regions. Depending on the genome assemblies and gene predictions, 30% of all SINE copies are associated with genes, particularly frequent in introns and untranslated regions (UTRs). The close association with genes is family specific. More than 10% of all genes annotated in the Solanaceae species investigated contain at least one SINE insertion, and we found genes harbouring up to 16 SINE copies. We demonstrate the involvement of SINEs in gene and genome evolution including the donation of splice sites, start and stop codons and exons to genes, enlargement of introns and UTRs, generation of tandem-like duplications and transduction of adjacent sequence regions. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

PubMed Central

Fu, X Y; Colgan, J D; Manley, J L

1988-01-01

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells. Images PMID:2851720

Systems analysis of cis-regulatory motifs in C4 photosynthesis genes using maize and rice leaf transcriptomic data during a process of de-etiolation

PubMed Central

Xu, Jiajia; Bräutigam, Andrea; Weber, Andreas P. M.; Zhu, Xin-Guang

2016-01-01

Identification of potential cis-regulatory motifs controlling the development of C4 photosynthesis is a major focus of current research. In this study, we used time-series RNA-seq data collected from etiolated maize and rice leaf tissues sampled during a de-etiolation process to systematically characterize the expression patterns of C4-related genes and to further identify potential cis elements in five different genomic regions (i.e. promoter, 5′UTR, 3′UTR, intron, and coding sequence) of C4 orthologous genes. The results demonstrate that although most of the C4 genes show similar expression patterns, a number of them, including chloroplast dicarboxylate transporter 1, aspartate aminotransferase, and triose phosphate transporter, show shifted expression patterns compared with their C3 counterparts. A number of conserved short DNA motifs between maize C4 genes and their rice orthologous genes were identified not only in the promoter, 5′UTR, 3′UTR, and coding sequences, but also in the introns of core C4 genes. We also identified cis-regulatory motifs that exist in maize C4 genes and also in genes showing similar expression patterns as maize C4 genes but that do not exist in rice C3 orthologs, suggesting a possible recruitment of pre-existing cis-elements from genes unrelated to C4 photosynthesis into C4 photosynthesis genes during C4 evolution. PMID:27436282

Antisense oligonucleotides effectively inhibit the co-transcriptional splicing of a Candida group I intron in vitro and in vivo: Implications for antifungal therapeutics.

PubMed

Zhang, Libin; Leibowitz, Michael J; Zhang, Yi

2009-02-18

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3-P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

Genomic Structure of an Economically Important Cyanobacterium, Arthrospira (Spirulina) platensis NIES-39

PubMed Central

Fujisawa, Takatomo; Narikawa, Rei; Okamoto, Shinobu; Ehira, Shigeki; Yoshimura, Hidehisa; Suzuki, Iwane; Masuda, Tatsuru; Mochimaru, Mari; Takaichi, Shinichi; Awai, Koichiro; Sekine, Mitsuo; Horikawa, Hiroshi; Yashiro, Isao; Omata, Seiha; Takarada, Hiromi; Katano, Yoko; Kosugi, Hiroki; Tanikawa, Satoshi; Ohmori, Kazuko; Sato, Naoki; Ikeuchi, Masahiko; Fujita, Nobuyuki; Ohmori, Masayuki

2010-01-01

A filamentous non-N2-fixing cyanobacterium, Arthrospira (Spirulina) platensis, is an important organism for industrial applications and as a food supply. Almost the complete genome of A. platensis NIES-39 was determined in this study. The genome structure of A. platensis is estimated to be a single, circular chromosome of 6.8 Mb, based on optical mapping. Annotation of this 6.7 Mb sequence yielded 6630 protein-coding genes as well as two sets of rRNA genes and 40 tRNA genes. Of the protein-coding genes, 78% are similar to those of other organisms; the remaining 22% are currently unknown. A total 612 kb of the genome comprise group II introns, insertion sequences and some repetitive elements. Group I introns are located in a protein-coding region. Abundant restriction-modification systems were determined. Unique features in the gene composition were noted, particularly in a large number of genes for adenylate cyclase and haemolysin-like Ca2+-binding proteins and in chemotaxis proteins. Filament-specific genes were highlighted by comparative genomic analysis. PMID:20203057

A domesticated transposon mediates the effects of a single-nucleotide polymorphism responsible for enhanced muscle growth.

PubMed

Butter, Falk; Kappei, Dennis; Buchholz, Frank; Vermeulen, Michiel; Mann, Matthias

2010-04-01

Single-nucleotide polymorphisms (SNPs) in the regulatory regions of the genome can have a profound impact on phenotype. The G3072A polymorphism in intron 3 of insulin-like growth factor 2 (IGF2) is implicated in higher muscle content and reduced fat in European pigs and is bound by a putative repressor. Here, we identify this repressor--which we call muscle growth regulator (MGR)--by using a DNA protein interaction screen based on quantitative mass spectrometry. MGR has a bipartite nuclear localization signal, two BED-type zinc fingers and is highly conserved between placental mammals. Surprisingly, the gene is located in an intron and belongs to the hobo-Ac-Tam3 transposase superfamily, suggesting regulatory use of a formerly parasitic element. In transactivation assays, MGR differentially represses the expression of the two SNP variants. Knockdown of MGR in C2C12 myoblast cells upregulates Igf2 expression and mild overexpression retards growth. Thus, MGR is the repressor responsible for enhanced muscle growth in the IGF2 G3072A polymorphism in commercially bred pigs.

Alternative Splicing as a Target for Cancer Treatment.

PubMed

Martinez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Anaya Ruiz, Maricruz; Monjaraz-Guzman, Eduardo; Martinez-Contreras, Rebeca

2018-02-11

Alternative splicing is a key mechanism determinant for gene expression in metazoan. During alternative splicing, non-coding sequences are removed to generate different mature messenger RNAs due to a combination of sequence elements and cellular factors that contribute to splicing regulation. A different combination of splicing sites, exonic or intronic sequences, mutually exclusive exons or retained introns could be selected during alternative splicing to generate different mature mRNAs that could in turn produce distinct protein products. Alternative splicing is the main source of protein diversity responsible for 90% of human gene expression, and it has recently become a hallmark for cancer with a full potential as a prognostic and therapeutic tool. Currently, more than 15,000 alternative splicing events have been associated to different aspects of cancer biology, including cell proliferation and invasion, apoptosis resistance and susceptibility to different chemotherapeutic drugs. Here, we present well established and newly discovered splicing events that occur in different cancer-related genes, their modification by several approaches and the current status of key tools developed to target alternative splicing with diagnostic and therapeutic purposes.

Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

PubMed

Rodríguez-Martín, Carlos; Cidre, Florencia; Fernández-Teijeiro, Ana; Gómez-Mariano, Gema; de la Vega, Leticia; Ramos, Patricia; Zaballos, Ángel; Monzón, Sara; Alonso, Javier

2016-05-01

Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

Angiotensin-converting enzyme gene insertion/deletion polymorphism studies in Asian Indian pregnant women biochemically identifies gestational diabetes mellitus.

PubMed

Khan, Imran A; Jahan, Parveen; Hasan, Qurratulain; Rao, Pragna

2014-12-01

Gestational diabetes mellitus (GDM) is defined as glucose intolerance first recognized during pregnancy. Insertion/deletion (I/D) polymorphism of a 287 bp Alu repetitive sequence in intron 16 of the angiotensin-converting enzyme (ACE) gene has been widely investigated in Asian Indian populations with different ethnic origins. The present study examined possible association between I/D polymorphism of the ACE gene and GDM in Asian Indian pregnant women. A total of 200 pregnant women (100 GDM and 100 non-GDM) were recruited in this study and I/D polymorphism of a 287 bp Alu1 element inside intron 16 of the ACE gene was examined by polymerase chain reaction (PCR)-based gel electrophoresis. The distribution of the variants like II, ID, and DD genotypes of ACE gene showed differences between normal GDM versus non-GDM subjects, and the frequency of the ID+ DD Vs II genotype was significant (p=0.0002) in the GDM group. ACE gene polymorphism was associated with GDM in Asian Indian pregnant women. © The Author(s) 2013.

Optimization of a novel biophysical model using large scale in vivo antisense hybridization data displays improved prediction capabilities of structurally accessible RNA regions

PubMed Central

Vazquez-Anderson, Jorge; Mihailovic, Mia K.; Baldridge, Kevin C.; Reyes, Kristofer G.; Haning, Katie; Cho, Seung Hee; Amador, Paul; Powell, Warren B.

2017-01-01

Abstract Current approaches to design efficient antisense RNAs (asRNAs) rely primarily on a thermodynamic understanding of RNA–RNA interactions. However, these approaches depend on structure predictions and have limited accuracy, arguably due to overlooking important cellular environment factors. In this work, we develop a biophysical model to describe asRNA–RNA hybridization that incorporates in vivo factors using large-scale experimental hybridization data for three model RNAs: a group I intron, CsrB and a tRNA. A unique element of our model is the estimation of the availability of the target region to interact with a given asRNA using a differential entropic consideration of suboptimal structures. We showcase the utility of this model by evaluating its prediction capabilities in four additional RNAs: a group II intron, Spinach II, 2-MS2 binding domain and glgC 5΄ UTR. Additionally, we demonstrate the applicability of this approach to other bacterial species by predicting sRNA–mRNA binding regions in two newly discovered, though uncharacterized, regulatory RNAs. PMID:28334800

A CT-rich haplotype in intron 4 of SNCA confers risk for Lewy body pathology in Alzheimer’s disease and affects SNCA expression

PubMed Central

Lutz, Michael W.; Saul, Robert; Linnertz, Colton; Glenn, Omolara-Chinue; Roses, Allen D.; Chiba-Falek, Ornit

2015-01-01

INTRODUCTION We recently showed that tagging-SNPs across the SNCA locus were significantly associated with increased risk for LB pathology in AD cases. However, the actual genetic variant(s) that underlie the observed associations remain elusive. METHODS We used a bioinformatics algorithm to catalogue Structural-Variants in a region of SNCA-intron4, followed by phased-sequencing. We performed a genetic-association analysis in autopsy series of LBV/AD cases compared with AD-only controls. We investigated the biological functions by expression analysis using temporal-cortex samples. RESULTS We identified four distinct haplotypes within a highly-polymorphic-low-complexity CT-rich region. We showed that a specific haplotype conferred risk to develop LBV/AD. We demonstrated that the CT-rich site acts as an enhancer element, where the risk haplotype was significantly associated with elevated levels of SNCA-mRNA. DISCUSSION We have discovered a novel haplotype in a CT-rich region in SNCA that contributes to LB pathology in AD patients, possibly via cis-regulation of the gene expression. PMID:26079410

Moving finite elements in 2-D

NASA Technical Reports Server (NTRS)

Gelinas, R. J.; Doss, S. K.; Vajk, J. P.; Djomehri, J.; Miller, K.

1983-01-01

The mathematical background regarding the moving finite element (MFE) method of Miller and Miller (1981) is discussed, taking into account a general system of partial differential equations (PDE) and the amenability of the MFE method in two dimensions to code modularization and to semiautomatic user-construction of numerous PDE systems for both Dirichlet and zero-Neumann boundary conditions. A description of test problem results is presented, giving attention to aspects of single square wave propagation, and a solution of the heat equation.

Transposable elements as a molecular evolutionary force

NASA Technical Reports Server (NTRS)

Fedoroff, N. V.

1999-01-01

This essay addresses the paradoxes of the complex and highly redundant genomes. The central theses developed are that: (1) the distinctive feature of complex genomes is the existence of epigenetic mechanisms that permit extremely high levels of both tandem and dispersed redundancy; (2) the special contribution of transposable elements is to modularize the genome; and (3) the labilizing forces of recombination and transposition are just barely contained, giving a dynamic genetic system of ever increasing complexity that verges on the chaotic.

Ice Accretion and Performance Degradation Calculations with LEWICE/NS

NASA Technical Reports Server (NTRS)

Potapczuk, Mark G.; Al-Khalil, Kamel M.; Velazquez, Matthew T.

1993-01-01

The LEWICE ice accretion computer code has been extended to include the solution of the two-dimensional Navier-Stokes equations. The code is modular and contains separate stand-alone program elements that create a grid, calculate the flow field parameters, calculate the droplet trajectory paths, determine the amount of ice growth, calculate aeroperformance changes, and plot results. The new elements of the code are described. Calculated results are compared to experiment for several cases, including both ice shape and drag rise.

Sequence Variation of the tRNALeu Intron as a Marker for Genetic Diversity and Specificity of Symbiotic Cyanobacteria in Some Lichens

PubMed Central

Paulsrud, Per; Lindblad, Peter

1998-01-01

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNALeu (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNALeu (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNALeu (UAA) intron can be of great value when examining cyanobacterial diversity. PMID:9435083

Identification and characterization of cell-specific enhancer elements for the mouse ETF/Tead2 gene.

PubMed

Tanoue, Y; Yasunami, M; Suzuki, K; Ohkubo, H

2001-12-21

We have identified and characterized by transient transfection assays the cell-specific 117-bp enhancer sequence in the first intron of the mouse ETF (Embryonic TEA domain-containing factor)/Tead2 gene required for transcriptional activation in ETF/Tead2 gene-expressing cells, such as P19 cells. The 117-bp enhancer contains one GC-rich sequence (5'-GGGGCGGGG-3'), termed the GC box, and two tandemly repeated GA-rich sequences (5'-GGGGGAGGGG-3'), termed the proximal and distal GA elements. Further analyses, including transfection studies and electrophoretic mobility shift assays using a series of deletion and mutation constructs, indicated that Sp1, a putative activator, may be required to predominate over its competition with another unknown putative repressor, termed the GA element-binding factor, for binding to both the GC box, which overlapped with the proximal GA element, and the distal GA element in the 117-bp sequence in order to achieve a full enhancer activity. We also discuss a possible mechanism underlying the cell-specific enhancer activity of the 117-bp sequence.

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology.

PubMed

Hernanz-Koers, Miguel; Gandía, Mónica; Garrigues, Sandra; Manzanares, Paloma; Yenush, Lynne; Orzaez, Diego; Marcos, Jose F

2018-07-01

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for Hyg R or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community. Copyright © 2018 Elsevier Inc. All rights reserved.

Three Group-I introns in 18S rDNA of Endosymbiotic Algae of Paramecium bursaria from Japan

NASA Astrophysics Data System (ADS)

Hoshina, Ryo; Kamako, Shin-ichiro; Imamura, Nobutaka

2004-08-01

In the nuclear encoded small subunit ribosomal DNA (18S rDNA) of symbiotic alga of Paramecium bursaria (F36 collected in Japan) possesses three intron-like insertions (Hoshina et al., unpubl. data, 2003). The present study confirmed these exact lengths and insertion sites by reverse transcription-PCR. Two of them were inserted at Escherichia coli 16S rRNA genic position 943 and 1512 that are frequent intron insertion positions, but another insertion position (nearly 1370) was the first finding. Their secondary structures suggested they belong to Group-I intron; one belongs to subgroup IE, others belong to subgroup IC1. Similarity search indicated these introns are ancestral ones.

DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic program in calcific aortic valve disease.

PubMed

Mkannez, Ghada; Gagné-Ouellet, Valérie; Nsaibia, Mohamed Jalloul; Boulanger, Marie-Chloé; Rosa, Mickael; Argaud, Deborah; Hadji, Fayez; Gaudreault, Nathalie; Rhéaume, Gabrielle; Bouchard, Luigi; Bossé, Yohan; Mathieu, Patrick

2018-05-02

Calcific aortic valve disease (CAVD) is characterized by the osteogenic transition of valve interstitial cells (VICs). In CAVD, lysophosphatidic acid (LysoPA), a lipid mediator with potent osteogenic activity, is produced in the aortic valve (AV) and is degraded by membrane-associated phospholipid phosphatases (PLPPs). We thus hypothesized that a dysregulation of PLPPs could participate to the osteogenic reprograming of VICs during CAVD. The expression of PLPPs was examined in human control and mineralized AVs and comprehensive analyses were performed to document the gene regulation and impact of PLPPs on the osteogenic transition of VICs. We found that PLPP3 gene and enzymatic activity were downregulated in mineralized AVs. Multidimensional gene profiling in 21 human AVs showed that expression of PLPP3 was inversely correlated with the level of 5-methylcytosine (5meC) located in an intronic mammalian interspersed repeat element (MIR). Bisulfite pyrosequencing in a larger series of 67 AVs confirmed that 5meC in intron 1 was increased by 2.2-fold in CAVD compared to control AVs. In isolated cells, epigenome editing with CRISPR-Cas9 system containing a deficient Cas9 fused with DNA methyltransferase (dCas9-DNMT) was used to increase 5meC in the intronic enhancer and showed that it reduced significantly the expression of PLPP3. Knockdown experiments showed that lower expression of PLPP3 in VICs promotes an osteogenic program. DNA methylation of a MIR-based enhancer downregulates the expression of PLPP3 and promotes the mineralization of the AV.

Genomic organization of the human gene (CA5) and pseudogene for mitochondrial carbonic anhydrase V and their localization to chromosomes 16q and 16p

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

Nagao, Yoshiro; Sly, W.S.; Batanian, J.R.

1995-08-10

Carbonic anhydrase V (CA V) is expressed in mitochondrial matrix in liver and several other tissues. It is of interest for its putative roles in providing bicarbonate to carbamoyl phosphate synthetase for ureagenesis and to pyruvate carboxylase for gluconeogenesis and its possible importance in explaining certain inherited metabolic disorders with hyperammonemia and hypoglycemia. Following the recent characterization of the cDNA for human CA V, we report the isolation of the human gene from two {lambda} genomic libraries and its characterization. The CA V gene (CA5) is approximately 50 kb long and contains 7 exons and 6 introns. The exon-intron boundariesmore » are found in positions identical to those determined for the previously described CA II, CA III, and CA VII genes. Like the CA VII gene, CA5 does not contain typical TATA and CAAT promoter elements in the 5{prime} flanking region but does contain a TTTAA sequence 147 nucleotides upstream of the initiation codon. CA5 also contains a 12-bp GT-rich segment beginning 13 bp downstream of the polyadenylation signal in the 3{prime} untranslated region of exon 7. FISH analysis allowed CA5 to be assigned to chromosome 16q24.3. An unprocessed pseudogene containing sequence homologous to exons 3-7 and introns 3-6 was also isolated and was assigned by FISH analysis to chromosome 16p11.2-p12. 22 refs., 4 figs., 1 tab.« less

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

PubMed Central

Gleitsman, Kristin R.

2014-01-01

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs. PMID:25246656

Isolation and Identification of Gene-Specific MicroRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2018-01-01

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducing RNA interference (RNAi)-related gene silencing, we have developed an intronic miRNA expression system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hence successfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanism has been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA so obtained is transcribed by type-II RNA polymerases, coexpressed within a primary gene transcript, and then excised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII) endonucleases further process the spliced introns into mature miRNAs. Using this intronic miRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitro but also in zebrafishes, chicken embryos, and adult mice in vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design various intronic pre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

Modular and Reusable Power System Design for the BRRISON Balloon Telescope

NASA Astrophysics Data System (ADS)

Truesdale, Nicholas A.

High altitude balloons are emerging as low-cost alternatives to orbital satellites in the field of telescopic observation. The near-space environment of balloons allows optics to perform near their diffraction limit. In practice, this implies that a telescope similar to the Hubble Space Telescope could be flown for a cost of tens of millions as opposed to billions. While highly feasible, the design of a balloon telescope to rival Hubble is limited by funding. Until a prototype is proven and more support for balloon science is gained, projects remain limited in both hardware costs and man hours. Thus, to effectively create and support balloon payloads, engineering designs must be efficient, modular, and if possible reusable. This thesis focuses specifically on a modular power system design for the BRRISON comet-observing balloon telescope. Time- and cost-saving techniques are developed that can be used for future missions. A modular design process is achieved through the development of individual circuit elements that span a wide range of capabilities. Circuits for power conversion, switching and sensing are designed to be combined in any configuration. These include DC-DC regulators, MOSFET drivers for switching, isolated switches, current sensors and voltage sensing ADCs. Emphasis is also given to commercially available hardware. Pre-fabricated DC-DC converters and an Arduino microcontroller simplify the design process and offer proven, cost-effective performance. The design of the BRRISON power system is developed from these low-level circuits elements. A board for main power distribution supports the majority of flight electronics, and is extensible to additional hardware in future applications. An ATX computer power supply is developed, allowing the use of a commercial ATX motherboard as the flight computer. The addition of new capabilities is explored in the form of a heater control board. Finally, the power system as a whole is described, and its overall performance analyzed. The success of the BRRISON power system during testing and flight proves its utility, both for BRRISON and for future balloon telescopes.

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

PubMed

Horiuchi, Keiko; Perez-Cerezales, Serafín; Papasaikas, Panagiotis; Ramos-Ibeas, Priscila; López-Cardona, Angela Patricia; Laguna-Barraza, Ricardo; Fonseca Balvís, Noelia; Pericuesta, Eva; Fernández-González, Raul; Planells, Benjamín; Viera, Alberto; Suja, Jose Angel; Ross, Pablo Juan; Alén, Francisco; Orio, Laura; Rodriguez de Fonseca, Fernando; Pintado, Belén; Valcárcel, Juan; Gutiérrez-Adán, Alfonso

2018-04-03

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

PubMed Central

De Wachter, R; Neefs, J M; Goris, A; Van de Peer, Y

1992-01-01

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing. PMID:1561081

Intronic L1 Retrotransposons and Nested Genes Cause Transcriptional Interference by Inducing Intron Retention, Exonization and Cryptic Polyadenylation

PubMed Central

Kaer, Kristel; Branovets, Jelena; Hallikma, Anni; Nigumann, Pilvi; Speek, Mart

2011-01-01

Background Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown. Methodology/Principal Findings Using in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3′ ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs) and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals. Conclusions/Significance Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression. PMID:22022525

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

PubMed Central

Edwards, Thomas E.; Phan, Isabelle; Abendroth, Jan; Dieterich, Shellie H.; Masoudi, Amir; Guo, Wenjin; Hewitt, Stephen N.; Kelley, Angela; Leibly, David; Brittnacher, Mitch J.; Staker, Bart L.; Miller, Samuel I.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

2010-01-01

Background Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region. Methodology/Principal Findings Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs. Conclusions/Significance The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics. PMID:20862217

Combining analysis with optimization at Langley Research Center. An evolutionary process

NASA Technical Reports Server (NTRS)

Rogers, J. L., Jr.

1982-01-01

The evolutionary process of combining analysis and optimization codes was traced with a view toward providing insight into the long term goal of developing the methodology for an integrated, multidisciplinary software system for the concurrent analysis and optimization of aerospace structures. It was traced along the lines of strength sizing, concurrent strength and flutter sizing, and general optimization to define a near-term goal for combining analysis and optimization codes. Development of a modular software system combining general-purpose, state-of-the-art, production-level analysis computer programs for structures, aerodynamics, and aeroelasticity with a state-of-the-art optimization program is required. Incorporation of a modular and flexible structural optimization software system into a state-of-the-art finite element analysis computer program will facilitate this effort. This effort results in the software system used that is controlled with a special-purpose language, communicates with a data management system, and is easily modified for adding new programs and capabilities. A 337 degree-of-freedom finite element model is used in verifying the accuracy of this system.

Progress on Fault Mechanisms for Gear Transmissions in Coal Cutting Machines: From Macro to Nano Models.

PubMed

Jiang, Yu; Zhang, Xiaogang; Zhang, Chao; Li, Zhixiong; Sheng, Chenxing

2017-04-01

Numerical modeling has been recognized as the dispensable tools for mechanical fault mechanism analysis. Techniques, ranging from macro to nano levels, include the finite element modeling boundary element modeling, modular dynamic modeling, nano dynamic modeling and so forth. This work firstly reviewed the progress on the fault mechanism analysis for gear transmissions from the tribological and dynamic aspects. Literature review indicates that the tribological and dynamic properties were separately investigated to explore the fault mechanism in gear transmissions. However, very limited work has been done to address the links between the tribological and dynamic properties and scarce researches have been done for coal cutting machines. For this reason, the tribo-dynamic coupled model was introduced to bridge the gap between the tribological and dynamic models in fault mechanism analysis for gear transmissions in coal cutting machines. The modular dynamic modeling and nano dynamic modeling techniques are expected to establish the links between the tribological and dynamic models. Possible future research directions using the tribo dynamic coupled model were summarized to provide potential references for researchers in the field.

Design of a Modular E-Core Flux Concentrating Axial Flux Machine: Preprint

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

Husain, Tausif; Sozer, Yilmaz; Husain, Iqbal

2015-08-24

In this paper a novel E-Core axial flux machine is proposed. The machine has a double-stator, single-rotor configuration with flux-concentrating ferrite magnets and pole windings across each leg of an E-Core stator. E-Core stators with the proposed flux-concentrating rotor arrangement result in better magnet utilization and higher torque density. The machine also has a modular structure facilitating simpler construction. This paper presents a single-phase and a three-phase version of the E-Core machine. Case studies for a 1.1-kW, 400-rpm machine for both the single-phase and three-phase axial flux machines are presented. The results are verified through 3D finite element analysis. facilitatingmore » simpler construction. This paper presents a single-phase and a three-phase version of the E-Core machine. Case studies for a 1.1-kW, 400-rpm machine for both the single-phase and three-phase axial flux machines are presented. The results are verified through 3D finite element analysis.« less

A flexible CAMAC based data system for Space Shuttle scientific instruments

NASA Technical Reports Server (NTRS)

Ehrmann, C. H.; Baker, R. G.; Smith, R. L.; Kaminski, T. J.

1979-01-01

An effort has been made within NASA to produce a low-cost modular system for implementation of Shuttle payloads based on the CAMAC standards for packaging and data transfer. A key element of such a modular system is a means for controlling the data system, collecting and processing the data for transmission to the ground, and issuing commands to the instrument either from the ground or based on the data collected. A description is presented of such a means based on a network of digital processors and CAMAC crate controllers, which allows for the implementation of instruments ranging from those requiring only a single CAMAC crate of functional modules and no data processing to ones requiring multiple crates and multiple data processors.

Design of a Modular E-Core Flux Concentrating Axial Flux Machine

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

Husain, Tausif; Sozer, Yilmaz; Husain, Iqbal

2015-09-02

In this paper a novel E-Core axial flux machine is proposed. The machine has a double stator-single rotor configuration with flux concentrating ferrite magnets, and pole windings across each leg of an E-Core stator. E-Core stators with the proposed flux-concentrating rotor arrangement result in better magnet utilization and higher torque density. The machine also has a modular structure facilitating simpler construction. This paper presents a single phase and a three-phase version of the E-Core machine. Case study for a 1.1 kW, 400 rpm machine for both the single phase and three-phase axial flux machine is presented. The results are verifiedmore » through 3D finite element analysis.« less

Position reporting system using small satellites

NASA Technical Reports Server (NTRS)

Pavesi, B.; Rondinelli, G.; Graziani, F.

1990-01-01

A system able to provide position reporting and monitoring services for mobile applications represents a natural complement to the Global Positioning System (GPS) navigation system. The system architecture is defined on the basis of the communications requirements derived by user needs, allowing maximum flexibility in the use of channel capacity, and a very simple and low cost terminal. The payload is sketched, outlining the block modularity and the use of qualified hardware. The global system capacity is also derived. The spacecraft characteristics are defined on the basis of the payload requirements. A small bus optimized for Ariane IV, Delta II vehicles and based on the modularity concept is presented. The design takes full advantage of each launcher with a common basic bus or bus elements for a mass production.

BeamDyn: A High-Fidelity Wind Turbine Blade Solver in the FAST Modular Framework: Preprint

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

Wang, Q.; Sprague, M.; Jonkman, J.

2015-01-01

BeamDyn, a Legendre-spectral-finite-element implementation of geometrically exact beam theory (GEBT), was developed to meet the design challenges associated with highly flexible composite wind turbine blades. In this paper, the governing equations of GEBT are reformulated into a nonlinear state-space form to support its coupling within the modular framework of the FAST wind turbine computer-aided engineering (CAE) tool. Different time integration schemes (implicit and explicit) were implemented and examined for wind turbine analysis. Numerical examples are presented to demonstrate the capability of this new beam solver. An example analysis of a realistic wind turbine blade, the CX-100, is also presented asmore » validation.« less

A modular solid state detector for measuring high energy heavy ion fragmentation near the beam axis

NASA Technical Reports Server (NTRS)

Zeitlin, C. J.; Frankel, K. A.; Gong, W.; Heilbronn, L.; Lampo, E. J.; Leres, R.; Miller, J.; Schimmerling, W.

1994-01-01

A multi-element solid state detector has been designed to measure fluences of fragments produced near the beam axis by high energy heavy ion beams in thick targets. The detector is compact and modular, so as to be readily reconfigured according to the range of fragment charges and energies to be measured. Preamplifier gain settings and detector calibrations are adjustable remotely under computer control. We describe the central detector, its associated detectors and electronics, triggering scheme, data acquisition and particle identification techniques, illustrated by data taken with 600 MeV/u 56Fe beams and thick polyethylene targets at the LBL Bevalac. The applications of this work to space radiation protection are discussed.

A pancreas specificity results from the combination of polyomavirus and Moloney murine leukemia virus enhancer.

PubMed Central

Rochford, R; Campbell, B A; Villarreal, L P

1987-01-01

An infectious recombinant polyomavirus was constructed in which a regulatory region of its genome, the B enhancer region (nucleotides 5128-5265) has been replaced with the 72- or 73-base-pair repeat enhancer from the Moloney murine leukemia virus genome. We show that this recombinant polyomavirus displays a strong tissue specificity for the pancreas of mice. This organ was not permissive for either the parental polyomavirus, which is predominantly kidney and salivary gland specific, or the Moloney murine leukemia virus, which is lymphotropic. This result indicated that tissue specificity can be achieved by a combination of apparently modular elements. Some of the implications of a modular mechanism of tissue specificity are considered. Images PMID:3025873

Modular System to Enable Extravehicular Activity

NASA Technical Reports Server (NTRS)

Sargusingh, Miriam J.

2012-01-01

The ability to perform extravehicular activity (EVA), both human and robotic, has been identified as a key component to space missions to support such operations as assembly and maintenance of space systems (e.g. construction and maintenance of the International Space Station), and unscheduled activities to repair an element of the transportation and habitation systems that can only be accessed externally and via unpressurized areas. In order to make human transportation beyond lower Earth orbit (LEO) practical, efficiencies must be incorporated into the integrated transportation systems to reduce system mass and operational complexity. Affordability is also a key aspect to be considered in space system development; this could be achieved through commonality, modularity and component reuse. Another key aspect identified for the EVA system was the ability to produce flight worthy hardware quickly to support early missions and near Earth technology demonstrations. This paper details a conceptual architecture for a modular EVA system that would meet these stated needs for EVA capability that is affordable, and that could be produced relatively quickly. Operational concepts were developed to elaborate on the defined needs, and to define the key capabilities, operational and design constraints, and general timelines. The operational concept lead to a high level design concept for a module that interfaces with various space transportation elements and contains the hardware and systems required to support human and telerobotic EVA; the module would not be self-propelled and would rely on an interfacing element for consumable resources. The conceptual architecture was then compared to EVA Systems used in the Space Shuttle Orbiter, on the International Space Station to develop high level design concepts that incorporate opportunities for cost savings through hardware reuse, and quick production through the use of existing technologies and hardware designs. An upgrade option was included to make use of the developing suit port technologies.

PIECE 2.0: an update for the plant gene structure comparison and evolution database

USDA-ARS?s Scientific Manuscript database

PIECE (Plant Intron Exon Comparision and Evolution) is a web-accessible database that houses intron and exon information of plant genes. PIECE serves as a resource for biologists interested in comparing intron-exon organization and provides valuable insights into the evolution of gene structure in ...

Functional comparison of three transformer gene introns regulating conditional female lethality

USDA-ARS?s Scientific Manuscript database

The trasformer gene plays a critical role in the sex determination pathways of many insects. We cloned two transformer gene introns from Anastrepha suspensa, the Caribbean fruit fly. These introns have sequences that putatively have a role in sex-specific splicing patterns that affect sex determinat...

Choosing and Using Introns in Molecular Phylogenetics

PubMed Central

Creer, Simon

2007-01-01

Introns are now commonly used in molecular phylogenetics in an attempt to recover gene trees that are concordant with species trees, but there are a range of genomic, logistical and analytical considerations that are infrequently discussed in empirical studies that utilize intron data. This review outlines expedient approaches for locus selection, overcoming paralogy problems, recombination detection methods and the identification and incorporation of LVHs in molecular systematics. A range of parsimony and Bayesian analytical approaches are also described in order to highlight the methods that can currently be employed to align sequences and treat indels in subsequent analyses. By covering the main points associated with the generation and analysis of intron data, this review aims to provide a comprehensive introduction to using introns (or any non-coding nuclear data partition) in contemporary phylogenetics. PMID:19461984

Comparative Analysis of Four Calypogeia Species Revealed Unexpected Change in Evolutionarily-Stable Liverwort Mitogenomes

PubMed Central

Ślipiko, Monika; Buczkowska-Chmielewska, Katarzyna; Bączkiewicz, Alina; Szczecińska, Monika; Sawicki, Jakub

2017-01-01

Liverwort mitogenomes are considered to be evolutionarily stable. A comparative analysis of four Calypogeia species revealed differences compared to previously sequenced liverwort mitogenomes. Such differences involve unexpected structural changes in the two genes, cox1 and atp1, which have lost three and two introns, respectively. The group I introns in the cox1 gene are proposed to have been lost by two-step localized retroprocessing, whereas one-step retroprocessing could be responsible for the disappearance of the group II introns in the atp1 gene. These cases represent the first identified losses of introns in mitogenomes of leafy liverworts (Jungermanniopsida) contrasting the stability of mitochondrial gene order with certain changes in the gene content and intron set in liverworts. PMID:29257096

Bio—Cryptography: A Possible Coding Role for RNA Redundancy

NASA Astrophysics Data System (ADS)

Regoli, M.

2009-03-01

The RNA-Crypto System (shortly RCS) is a symmetric key algorithm to cipher data. The idea for this new algorithm starts from the observation of nature. In particular from the observation of RNA behavior and some of its properties. The RNA sequences have some sections called Introns. Introns, derived from the term "intragenic regions," are non-coding sections of precursor mRNA (pre-mRNA) or other RNAs, that are removed (spliced out of the RNA) before the mature RNA is formed. Once the introns have been spliced out of a pre-mRNA, the resulting mRNA sequence is ready to be translated into a protein. The corresponding parts of a gene are known as introns as well. The nature and the role of Introns in the pre-mRNA is not clear and it is under ponderous researches by biologists but, in our case, we will use the presence of Introns in the RNA-Crypto System output as a strong method to add chaotic non coding information and an unnecessary behavior in the access to the secret key to code the messages. In the RNA-Crypto System algorithm the introns are sections of the ciphered message with non-coding information as well as in the precursor mRNA.

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication.

PubMed

Wang, Hongtao; Li, Guisheng; Kwon, Woo-Saeng; Yang, Deok-Chun

2016-06-04

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to "Gopoong" and "K-1" were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information.

RNA chaperone StpA loosens interactions of the tertiary structure in the td group I intron in vivo

PubMed Central

Waldsich, Christina; Grossberger, Rupert; Schroeder, Renée

2002-01-01

Efficient splicing of the td group I intron in vivo is dependent on the ribosome. In the absence of translation, the pre-mRNA is trapped in nonnative-splicing-incompetent conformations. Alternatively, folding of the pre-mRNA can be promoted by the RNA chaperone StpA or by the group I intron-specific splicing factor Cyt-18. To understand the mechanism of action of RNA chaperones, we probed the impact of StpA on the structure of the td intron in vivo. Our data suggest that StpA loosens tertiary interactions. The most prominent structural change was the opening of the base triples, which are involved in the correct orientation of the two major intron core domains. In line with the destabilizing activity of StpA, splicing of mutant introns with a reduced structural stability is sensitive to StpA. In contrast, Cyt-18 strengthens tertiary contacts, thereby rescuing splicing of structurally compromised td mutants in vivo. Our data provide direct evidence for protein-induced conformational changes within catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the overall compactness of the td intron in vivo. PMID:12208852

Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

PubMed

Lunghi, Matteo; Spano, Furio; Magini, Alessandro; Emiliani, Carla; Carruthers, Vern B; Di Cristina, Manlio

2016-02-01

Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.

Two distinct promoters drive transcription of the human D1A dopamine receptor gene.

PubMed

Lee, S H; Minowa, M T; Mouradian, M M

1996-10-11

The human D1A dopamine receptor gene has a GC-rich, TATA-less promoter located upstream of a small, noncoding exon 1, which is separated from the coding exon 2 by a 116-base pair (bp)-long intron. Serial 3'-deletions of the 5'-noncoding region of this gene, including the intron and 5'-end of exon 2, resulted in 80 and 40% decrease in transcriptional activity of the upstream promoter in two D1A-expressing neuroblastoma cell lines, SK-N-MC and NS20Y, respectively. To investigate the function of this region, the intron and 245 bp at the 5'-end of exon 2 were investigated. Transient expression analyses using various chloramphenicol acetyltransferase constructs showed that the transcriptional activity of the intron is higher than that of the upstream promoter by 12-fold in SK-N-MC cells and by 5.5-fold in NS20Y cells in an orientation-dependent manner, indicating that the D1A intron is a strong promoter. Primer extension and ribonuclease protection assays revealed that transcription driven by the intron promoter is initiated at the junction of intron and exon 2 and at a cluster of nucleotides located 50 bp downstream from this junction. The same transcription start sites are utilized by the chloramphenicol acetyltransferase constructs employed in transfections as well as by the D1A gene expressed within the human caudate. The relative abundance of D1A transcripts originating from the upstream promoter compared with those transcribed from the intron promoter is 1.5-2.9 times in SK-N-MC cells and 2 times in the human caudate. Transcript stability studies in SK-N-MC cells revealed that longer D1A mRNA molecules containing exon 1 are degraded 1.8 times faster than shorter transcripts lacking exon 1. Although gel mobility shift assay could not detect DNA-protein interaction at the D1A intron, competitive co-transfection using the intron as competitor confirmed the presence of trans-acting factors at the intron. These data taken together indicate that the human D1A gene has two functional TATA-less promoters, both in D1A expressing cultured neuroblastoma cells and in the human striatum.

The occurrence of spring forms in tetraploid Timopheevi wheat is associated with variation in the first intron of the VRN-A1 gene.

PubMed

Shcherban, Andrey Borisovich; Schichkina, Aleksandra Aleksandrovna; Salina, Elena Artemovna

2016-11-16

Triticum araraticum and Triticum timopheevii are tetraploid species of the Timopheevi group. The former includes both winter and spring forms with a predominance of winter forms, whereas T. timopheevii is considered a spring species. In order to clarify the origin of the spring growth habit in T. timopheevii, allelic variability of the VRN-1 gene was investigated in a set of accessions of both tetraploid species, together with the diploid species Ae. speltoides, presumed donor of the G genome to these tetraploids. The promoter region of the VRN-A1 locus in all studied tetraploid accessions of both T. araraticum and T. timopheevii represents the previously described allele VRN-A1f with a 50 bp deletion near the start codon. Three additional alleles were identified namely, VRN-A1f-del, VRN-A1f-ins and VRN-A1f-del/ins, which contained large mutations in the first (1 st ) intron of VRN-A1. The first allele, carrying a deletion of 2.7 kb in a central part of intron 1, occurred in a few accessions of T. araraticum and no accessions of T. timopheevii. The VRN-A1f-ins allele, containing the insertion of a 0.4 kb MITE element about 0.4 kb upstream from the start of intron 1, and allele VRN-A1f-del/ins having this insertion coupled with a deletion of 2.7 kb are characteristic only for T. timopheevii. Allelic variation at the VRN-G1 locus includes the previously described allele VRN-G1a (with the insertion of a 0.2 kb MITE in the promoter) found in a few accessions of both tetraploid species. We showed that alleles VRN-A1f-del and VRN-G1a have no association with the spring growth habit, while in all accessions of T. timopheevii this habit was associated with the dominant VRN-A1f-ins and VRN-A1f-del/ins alleles. None of the Ae. speltoides accessions included in this study had changes in the promoter or 1 st intron regions of VRN-1 which might confer a spring growth habit. The VRN-1 promoter sequences analyzed herein and downloaded from databases have been used to construct a phylogram to assess the time of divergence of Ae. speltoides in relation to other wheat species. Among accessions of T. araraticum, the preferentially winter predecessor of T. timopheevii, two large mutations were found in both VRN-A1 and VRN-G1 loci (VRN-A1f-del and VRN-G1a) that were found to have no effect on vernalization requirements. Spring tetraploid T. timopheevii had one VRN-1 allele in common for two species (VRN-G1a), and two that were specific (VRN-A1f-ins, VRN-A1f-del/ins). The latter alleles include mutations in the 1 st intron of VRN-A1 and also share a 0.4 kb MITE insertion near the start of intron 1. We suggested that this insertion resulted in a spring growth habit in a progenitor of T. timopheevii which has probably been selected during subsequent domestication. The phylogram constructed on the basis of the VRN-1 promoter sequences confirmed the early divergence (~3.5 MYA) of the ancestor(s) of the B/G genomes from Ae. speltoides.

Convectively cooled electrical grid structure

DOEpatents

Paterson, J.A.; Koehler, G.W.

1980-11-10

Undesirable distortions of electrical grid conductors from thermal cycling are minimized and related problems such as unwanted thermionic emission and structural failure from overheating are avoided by providing for a flow of fluid coolant within each conductor. The conductors are secured at each end to separate flexible support elements which accommodate to individual longitudinal expansion and contraction of each conductor while resisting lateral displacements, the coolant flow preferably being directed into and out of each conductor through passages in the flexible support elements. The grid may have a modular or divided construction which facilitates manufacture and repairs.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria.

PubMed

Santamaria, Monica; Vicario, Saverio; Pappadà, Graziano; Scioscia, Gaetano; Scazzocchio, Claudio; Saccone, Cecilia

2009-06-16

A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers. The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries. After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals. The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode Initiative. The large scale investigation of Ascomycota mitochondrial introns performed through this tool, allowing to exclude the introns-rich sequences from the barcode candidates exploration, could be the first step towards a mitochondrial barcoding strategy for these organisms, similar to the standard approach employed in metazoans.

Thermal Characterization for a Modular 3-D Multichip Module

NASA Technical Reports Server (NTRS)

Fan, Mark S.; Plante, Jeannette; Shaw, Harry

2000-01-01

NASA Goddard Space Flight Center has designed a high-density modular 3-D multichip module (MCM) for future spaceflight use. This MCM features a complete modular structure, i.e., each stack can be removed from the package without damaging the structure. The interconnection to the PCB is through the Column Grid Array (CGA) technology. Because of its high-density nature, large power dissipation from multiple layers of circuitry is anticipated and CVD diamond films are used in the assembly for heat conduction enhancement. Since each stacked layer dissipates certain amount of heat, designing effective heat conduction paths through each stack and balancing the heat dissipation within each stack for optimal thermal performance become a challenging task. To effectively remove the dissipated heat from the package, extensive thermal analysis has been performed with finite element methods. Through these analyses, we are able to improve the thermal design and increase the total wattage of the package for maximum electrical performance. This paper provides details on the design-oriented thermal analysis and performance enhancement. It also addresses issues relating to contact thermal resistance between the diamond film and the metallic heat conduction paths.

Hardware-Abbildung eines videobasierten Verfahrens zur echtzeitfähigen Auswertung von Winkelhistogrammen auf eine modulare Coprozessor-Architektur

NASA Astrophysics Data System (ADS)

Flatt, H.; Tarnowsky, A.; Blume, H.; Pirsch, P.

2010-10-01

Dieser Beitrag behandelt die Abbildung eines videobasierten Verfahrens zur echtzeitfähigen Auswertung von Winkelhistogrammen auf eine modulare Coprozessor-Architektur. Die Architektur besteht aus mehreren dedizierten Recheneinheiten zur parallelen Verarbeitung rechenintensiver Bildverarbeitungsverfahren und ist mit einem RISC-Prozessor verbunden. Eine konfigurierbare Architekturerweiterung um eine Recheneinheit zur Auswertung von Winkelhistogrammen von Objekten ermöglicht in Verbindung mit dem RISC eine echtzeitfähige Klassifikation. Je nach Konfiguration sind für die Architekturerweiterung auf einem Xilinx Virtex-5-FPGA zwischen 3300 und 12 000 Lookup-Tables erforderlich. Bei einer Taktfrequenz von 100 MHz können unabhängig von der Bildauflösung pro Einzelbild in einem 25-Hz-Videodatenstrom bis zu 100 Objekte der Größe 256×256 Pixel analysiert werden. This paper presents the mapping of a video-based approach for real-time evaluation of angular histograms on a modular coprocessor architecture. The architecture comprises several dedicated processing elements for parallel processing of computation-intensive image processing tasks and is coupled with a RISC processor. A configurable architecture extension, especially a processing element for evaluating angular histograms of objects in conjunction with a RISC processor, provides a real-time classification. Depending on the configuration of the architecture extension, 3 300 to 12 000 look-up tables are required for a Xilinx Virtex-5 FPGA implementation. Running at a clock frequency of 100 MHz and independently of the image resolution per frame, 100 objects of size 256×256 pixels are analyzed in a 25 Hz video stream by the architecture.

Systems analysis of cis-regulatory motifs in C4 photosynthesis genes using maize and rice leaf transcriptomic data during a process of de-etiolation.

PubMed

Xu, Jiajia; Bräutigam, Andrea; Weber, Andreas P M; Zhu, Xin-Guang

2016-09-01

Identification of potential cis-regulatory motifs controlling the development of C4 photosynthesis is a major focus of current research. In this study, we used time-series RNA-seq data collected from etiolated maize and rice leaf tissues sampled during a de-etiolation process to systematically characterize the expression patterns of C4-related genes and to further identify potential cis elements in five different genomic regions (i.e. promoter, 5'UTR, 3'UTR, intron, and coding sequence) of C4 orthologous genes. The results demonstrate that although most of the C4 genes show similar expression patterns, a number of them, including chloroplast dicarboxylate transporter 1, aspartate aminotransferase, and triose phosphate transporter, show shifted expression patterns compared with their C3 counterparts. A number of conserved short DNA motifs between maize C4 genes and their rice orthologous genes were identified not only in the promoter, 5'UTR, 3'UTR, and coding sequences, but also in the introns of core C4 genes. We also identified cis-regulatory motifs that exist in maize C4 genes and also in genes showing similar expression patterns as maize C4 genes but that do not exist in rice C3 orthologs, suggesting a possible recruitment of pre-existing cis-elements from genes unrelated to C4 photosynthesis into C4 photosynthesis genes during C4 evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Spliceman2: a computational web server that predicts defects in pre-mRNA splicing.

PubMed

Cygan, Kamil Jan; Sanford, Clayton Hendrick; Fairbrother, William Guy

2017-09-15

Most pre-mRNA transcripts in eukaryotic cells must undergo splicing to remove introns and join exons, and splicing elements present a large mutational target for disease-causing mutations. Splicing elements are strongly position dependent with respect to the transcript annotations. In 2012, we presented Spliceman, an online tool that used positional dependence to predict how likely distant mutations around annotated splice sites were to disrupt splicing. Here, we present an improved version of the previous tool that will be more useful for predicting the likelihood of splicing mutations. We have added industry-standard input options (i.e. Spliceman now accepts variant call format files), which allow much larger inputs than previously available. The tool also can visualize the locations-within exons and introns-of sequence variants to be analyzed and the predicted effects on splicing of the pre-mRNA transcript. In addition, Spliceman2 integrates with RNAcompete motif libraries to provide a prediction of which trans -acting factors binding sites are disrupted/created and links out to the UCSC genome browser. In summary, the new features in Spliceman2 will allow scientists and physicians to better understand the effects of single nucleotide variations on splicing. Freely available on the web at http://fairbrother.biomed.brown.edu/spliceman2 . Website implemented in PHP framework-Laravel 5, PostgreSQL, Apache, and Perl, with all major browsers supported. william_fairbrother@brown.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Characterization of a Gene Encoding Clathrin Heavy Chain in Maize Up-Regulated by Salicylic Acid, Abscisic Acid and High Boron Supply

PubMed Central

Zeng, Mu-Heng; Liu, Sheng-Hong; Yang, Miao-Xian; Zhang, Ya-Jun; Liang, Jia-Yong; Wan, Xiao-Rong; Liang, Hong

2013-01-01

Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA, suggesting that ZmCHC1 gene may be involved in the SA signaling pathway in maize defense responses. The expressions of ZmCHC1 and ZmCHC2 genes in maize are down-regulated by azide or cold treatment, further revealing the energy requirement of CME and suggesting that CME in plants is sensitive to low temperatures. PMID:23880865

Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales.

PubMed

Palumbi, S R; Baker, C S

1994-05-01

Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.

Analysis of Claviceps africana and C. sorghi from India using AFLPs, EF-1alpha gene intron 4, and beta-tubulin gene intron 3.

PubMed

Tooley, Paul W; Bandyopadhyay, Ranajit; Carras, Marie M; Pazoutová, Sylvie

2006-04-01

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1alpha gene intron 4 and beta-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1alpha gene intron 4, the beta-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.

Cell type-specific termination of transcription by transposable element sequences.

PubMed

Conley, Andrew B; Jordan, I King

2012-09-30

Transposable elements (TEs) encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS) genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3' UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are evolutionarily young. The extent of transcription termination by TEs seen here, along with the preference for sense-oriented TE insertions to provide TTS, is consistent with the observed antisense orientation bias of human TEs.

Contact mechanics of modular metal-on-polyethylene total hip replacement under adverse edge loading conditions

PubMed Central

Hua, Xijin; Li, Junyan; Wang, Ling; Jin, Zhongmin; Wilcox, Ruth; Fisher, John

2014-01-01

Edge loading can negatively impact the biomechanics and long-term performance of hip replacements. Although edge loading has been widely investigated for hard-on-hard articulations, limited work has been conducted for hard-on-soft combinations. The aim of the present study was to investigate edge loading and its effect on the contact mechanics of a modular metal-on-polyethylene (MoP) total hip replacement (THR). A three-dimensional finite element model was developed based on a modular MoP bearing. Different cup inclination angles and head lateral microseparation were modelled and their effect on the contact mechanics of the modular MoP hip replacement were examined. The results showed that lateral microseparation caused loading of the head on the rim of the cup, which produced substantial increases in the maximum von Mises stress in the polyethylene liner and the maximum contact pressure on both the articulating surface and backside surface of the liner. Plastic deformation of the liner was observed under both standard conditions and microseparation conditions, however, the maximum equivalent plastic strain in the liner under microseparation conditions of 2000 µm was predicted to be approximately six times that under standard conditions. The study has indicated that correct positioning the components to avoid edge loading is likely to be important clinically even for hard-on-soft bearings for THR. PMID:25218504

OpenDrift - an open source framework for ocean trajectory modeling

NASA Astrophysics Data System (ADS)

Dagestad, Knut-Frode; Breivik, Øyvind; Ådlandsvik, Bjørn

2016-04-01

We will present a new, open source tool for modeling the trajectories and fate of particles or substances (Lagrangian Elements) drifting in the ocean, or even in the atmosphere. The software is named OpenDrift, and has been developed at Norwegian Meteorological Institute in cooperation with Institute of Marine Research. OpenDrift is a generic framework written in Python, and is openly available at https://github.com/knutfrode/opendrift/. The framework is modular with respect to three aspects: (1) obtaining input data, (2) the transport/morphological processes, and (3) exporting of results to file. Modularity is achieved through well defined interfaces between components, and use of a consistent vocabulary (CF conventions) for naming of variables. Modular input implies that it is not necessary to preprocess input data (e.g. currents, wind and waves from Eulerian models) to a particular file format. Instead "reader modules" can be written/used to obtain data directly from any original source, including files or through web based protocols (e.g. OPeNDAP/Thredds). Modularity of processes implies that a model developer may focus on the geophysical processes relevant for the application of interest, without needing to consider technical tasks such as reading, reprojecting, and colocating input data, rotation and scaling of vectors and model output. We will show a few example applications of using OpenDrift for predicting drifters, oil spills, and search and rescue objects.

Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron.

PubMed

Hardy, C M; Clark-Walker, G D

1991-07-01

The cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8,826 bp and contains five exons (termed E1-E5) totalling 1,602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1-1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5 a, and 1.5 b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5 a and K1 cox1.4/Sc cox1.5 b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.

Chloroplast genome expansion by intron multiplication in the basal psychrophilic euglenoid Eutreptiella pomquetensis

PubMed Central

Bennett, Matthew S.; Triemer, Richard E.; Preisfeld, Angelika

2017-01-01

Background Over the last few years multiple studies have been published showing a great diversity in size of chloroplast genomes (cpGenomes), and in the arrangement of gene clusters, in the Euglenales. However, while these genomes provided important insights into the evolution of cpGenomes across the Euglenales and within their genera, only two genomes were analyzed in regard to genomic variability between and within Euglenales and Eutreptiales. To better understand the dynamics of chloroplast genome evolution in early evolving Eutreptiales, this study focused on the cpGenome of Eutreptiella pomquetensis, and the spread and peculiarities of introns. Methods The Etl. pomquetensis cpGenome was sequenced, annotated and afterwards examined in structure, size, gene order and intron content. These features were compared with other euglenoid cpGenomes as well as those of prasinophyte green algae, including Pyramimonas parkeae. Results and Discussion With about 130,561 bp the chloroplast genome of Etl. pomquetensis, a basal taxon in the phototrophic euglenoids, was considerably larger than the two other Eutreptiales cpGenomes sequenced so far. Although the detected quadripartite structure resembled most green algae and plant chloroplast genomes, the gene content of the single copy regions in Etl. pomquetensis was completely different from those observed in green algae and plants. The gene composition of Etl. pomquetensis was extensively changed and turned out to be almost identical to other Eutreptiales and Euglenales, and not to P. parkeae. Furthermore, the cpGenome of Etl. pomquetensis was unexpectedly permeated by a high number of introns, which led to a substantially larger genome. The 51 identified introns of Etl. pomquetensis showed two major unique features: (i) more than half of the introns displayed a high level of pairwise identities; (ii) no group III introns could be identified in the protein coding genes. These findings support the hypothesis that group III introns are degenerated group II introns and evolved later. PMID:28852596

Integrated three-dimensional module heat exchanger for power electronics cooling

DOEpatents

Bennion, Kevin; Lustbader, Jason

2013-09-24

Embodiments discussed herein are directed to a power semiconductor packaging that removes heat from a semiconductor package through one or more cooling zones that are located in a laterally oriented position with respect to the semiconductor package. Additional embodiments are directed to circuit elements that are constructed from one or more modular power semiconductor packages.

A Virtual World of Visualization

NASA Technical Reports Server (NTRS)

1998-01-01

In 1990, Sterling Software, Inc., developed the Flow Analysis Software Toolkit (FAST) for NASA Ames on contract. FAST is a workstation based modular analysis and visualization tool. It is used to visualize and animate grids and grid oriented data, typically generated by finite difference, finite element and other analytical methods. FAST is now available through COSMIC, NASA's software storehouse.

The IGF1 small dog haplotype is derived from Middle Eastern grey wolves.

PubMed

Gray, Melissa M; Sutter, Nathan B; Ostrander, Elaine A; Wayne, Robert K

2010-02-24

A selective sweep containing the insulin-like growth factor 1 (IGF1) gene is associated with size variation in domestic dogs. Intron 2 of IGF1 contains a SINE element and single nucleotide polymorphism (SNP) found in all small dog breeds that is almost entirely absent from large breeds. In this study, we surveyed a large sample of grey wolf populations to better understand the ancestral pattern of variation at IGF1 with a particular focus on the distribution of the small dog haplotype and its relationship to the origin of the dog. We present DNA sequence data that confirms the absence of the derived small SNP allele in the intron 2 region of IGF1 in a large sample of grey wolves and further establishes the absence of a small dog associated SINE element in all wild canids and most large dog breeds. Grey wolf haplotypes from the Middle East have higher nucleotide diversity suggesting an origin there. Additionally, PCA and phylogenetic analyses suggests a closer kinship of the small domestic dog IGF1 haplotype with those from Middle Eastern grey wolves. The absence of both the SINE element and SNP allele in grey wolves suggests that the mutation for small body size post-dates the domestication of dogs. However, because all small dogs possess these diagnostic mutations, the mutations likely arose early in the history of domestic dogs. Our results show that the small dog haplotype is closely related to those in Middle Eastern wolves and is consistent with an ancient origin of the small dog haplotype there. Thus, in concordance with past archeological studies, our molecular analysis is consistent with the early evolution of small size in dogs from the Middle East.See associated opinion by Driscoll and Macdonald: http://jbiol.com/content/9/2/10.

The PROGINS polymorphism of the human progesterone receptor diminishes the response to progesterone.

PubMed

Romano, Andrea; Delvoux, Bert; Fischer, Dagmar-Christiane; Groothuis, Patrick

2007-02-01

The human progesterone receptor (PR) is a ligand-dependent transcription factor and two isoforms, (PRA and PRB), can be distinguished. PROGINS, a PR polymorphic variant, affects PRA and PRB and acts as a risk-modulating factor in several gynaecological disorders. Little is known about the functional consequences of this variant. Here, we characterise the properties of PROGINS with respect to transcription, mRNA maturation, protein activity and proliferation. PROGINS is characterised by a 320 bp PV/HS-1 Alu insertion in intron G and two point mutations, V660L in exon 4 and H770H (silent substitution) in exon 5. The Alu element contains a half oestrogen-response element/Sp1-binding site (Alu-ERE/Sp1), which acts as an in-cis intronic enhancer leading to increased transcription of the PROGINS allele in response to 17beta-oestradiol. Moreover, Alu insertions in the human genome are frequently methylated. Our data indicate that the PROGINS-Alu does not affect gene transcription due to DNA methylation. However, the Alu element reduced the stability of the PROGINS transcript compared with the CP allele and does not generate splice variants. The amino acid substitution (V600L) in exon 4 leads to differences in PR phosphorylation and degradation in the two PR variants upon ligand binding, most likely as a result of differences in the three-dimensional structures of the two PR variants. As a consequence, the PR-L660 (PROGINS) variant (1) displays decreased transactivation activity in a luciferase reporter system and (2) is less efficient in opposing cell proliferation in hamster ovarian cells expressing human PRA, when compared with the PR-V660 (most common variant). Taken together, our results indicate that the PROGINS variant of PR is less responsive to progestin compared with the most common PR because of (i) reduced amounts of gene transcript and (ii) decreased protein activity.

GBF1 differentially regulates CAT2 and PAD4 transcription to promote pathogen defense in Arabidopsis thaliana.

PubMed

Giri, Mrunmay K; Singh, Nidhi; Banday, Zeeshan Z; Singh, Vijayata; Ram, Hathi; Singh, Deepjyoti; Chattopadhyay, Sudip; Nandi, Ashis K

2017-09-01

G-BOX BINDING FACTOR 1 (GBF1) influences light-regulated seedling development in Arabidopsis, and inhibits CATALASE 2 (CAT2) expression during senescence. CAT2 functions as a scavenger of hydrogen peroxide. The role of GBF1 in the defense response is not known. We report here that GBF1 positively influences the defense against virulent and avirulent strains of Pseudomonas syringae. The gbf1 mutants are susceptible, whereas GBF1 over-expresser transgenic plants are resistant to bacterial pathogens. GBF1 negatively regulates pathogen-induced CAT2 expression and thereby positively regulates the hypersensitive response. In addition to CAT2 promoter, GBF1 binds to the G-box-like element present in the intron of PHYTOALEXIN DEFICIENT 4 (PAD4). This association of GBF1 with PAD4 intron is enhanced upon pathogenesis. GBF1 positively regulates PAD4 transcription in an intron-dependent manner. GBF1-mediated positive regulation of PAD4 expression is also evident in gbf1 mutant and GBF1 over-expression lines. Similar to pad4 mutants, pathogen-induced camalexin and salicylic acid (SA) accumulation, and expression of SA-inducible PATHOGENESIS RELATED1 (PR1) gene are compromised in the gbf1 mutant. Exogenous application of SA rescues the loss-of-defense phenotypes of gbf1 mutant. Thus, altogether, our results demonstrate that GBF1 is an important component of the plant defense response that functions upstream of SA accumulation and, by oppositely regulating CAT2 and PAD4, promotes disease resistance in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Regulation of Bach2 by the aryl hydrocarbon receptor as a mechanism for suppression of B-cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

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

De Abrew, K. Nadira; Phadnis, Ashwini S.; Genetics Program, Michigan State University, East Lansing, MI 48824

2011-04-15

Exposure to the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters B-cell differentiation and suppresses antibody production. Previous genomic studies in mouse B cells identified Bach2 as a direct target of the AHR. Bach2 is known to repress expression of Prdm1, a key transcription factor involved in B-cell differentiation, by binding to Maf elements (MAREs) in the regulatory regions of the gene. Chromatin immunoprecipitation followed by quantitative PCR in TCDD-treated lipopolysaccharide (LPS)-activated B cells showed increased binding of the AHR within the first intron in the Bach2 gene. The binding was further confirmed by electrophoretic mobility shift assay (EMSA). TCDDmore » also induced expression of Bach2 in activated as well as resting B cells from 2 to 24 h post-treatment in a time- and concentration-dependent manner. Expression of Prdm1 was decreased by TCDD at 24 h and was consistent with repression by Bach2. Increased DNA binding activity to the intron 5 MARE with increasing TCDD concentrations was observed by EMSA. Supershifts identified the presence of Bach2 in the DNA binding complex associated with the intron 5 MARE of Prdm1. Functional validation of the role of Bach2 in the suppression of B-cell differentiation by TCDD was performed using RNA interference (RNAi). Knockdown of Bach2 showed approximately 40% reversal in the TCDD-induced suppression of IgM secretion when compared to controls. The results suggest that the transcriptional regulation of Bach2 by the AHR is one of the mechanisms involved in the suppression of B-cell differentiation by TCDD.« less

Regulation of Bach2 by the Aryl-hydrocarbon Receptor as a Mechanism for Suppression of B-cell Differentiation by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

PubMed Central

De Abrew, K. Nadira; Phadnis, Ashwini S.; Crawford, Robert B.; Kaminski, Norbert E.; Thomas, Russell S.

2011-01-01

Exposure to the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), alters B cell differentiation and suppresses antibody production. Previous genomic studies in mouse B cells identified Bach2 as a direct target of the AHR. Bach2 is known to repress expression of Prdm1, a key transcription factor involved in B cell differentiation, by binding to Maf elements (MAREs) in the regulatory regions of the gene. Chromatin immunopreciptiation followed by quantitative PCR in TCDD-treated lipopolysaccharide (LPS)-activated B cells showed increased binding of the AHR within the first intron in the Bach2 gene. The binding was further confirmed by electrophoretic mobility shift assay (EMSA). TCDD also induced expression of Bach2 in activated as well as resting B cells from 2 to 24 h post-treatment in a time and concentration-dependent manner. Expression of Prdm1 was decreased by TCDD at 24 h and was consistent with repression by Bach2. Increased DNA binding activity to the intron 5 MARE with increasing TCDD concentration was observed by EMSA. Supershifts identified the presence of Bach2 in the DNA binding complex associated with the intron5 MARE of Prdm1. Functional validation of the role of Bach2 in the suppression of B-cell differentiation by TCDD was performed using RNAi. Knockdown of Bach2 showed approximately 40% reversal in the TCDD-induced suppression of IgM secretion when compared to controls. The results suggest that the transcriptional regulation of Bach2 by the AHR is one of the mechanisms involved in the suppression of B-cell differentiation by TCDD. PMID:21296099

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication

PubMed Central

Wang, Hongtao; Li, Guisheng; Kwon, Woo-Saeng; Yang, Deok-Chun

2016-01-01

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to “Gopoong” and “K-1” were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information. PMID:27271615

Quaternary arrangement of an active, native group II intron ribonucleoprotein complex revealed by small-angle X-ray scattering.

PubMed

Gupta, Kushol; Contreras, Lydia M; Smith, Dorie; Qu, Guosheng; Huang, Tao; Spruce, Lynn A; Seeholzer, Steven H; Belfort, Marlene; Van Duyne, Gregory D

2014-04-01

The stable ribonucleoprotein (RNP) complex formed between the Lactococcus lactis group II intron and its self-encoded LtrA protein is essential for the intron's genetic mobility. In this study, we report the biochemical, compositional, hydrodynamic and structural properties of active group II intron RNP particles (+A) isolated from its native host using a novel purification scheme. We employed small-angle X-ray scattering to determine the structural properties of these particles as they exist in solution. Using sucrose as a contrasting agent, we derived a two-phase quaternary model of the protein-RNA complex. This approach revealed that the spatial properties of the complex are largely defined by the RNA component, with the protein dimer located near the center of mass. A transfer RNA fusion engineered into domain II of the intron provided a distinct landmark consistent with this interpretation. Comparison of the derived +A RNP shape with that of the previously reported precursor intron (ΔA) particle extends previous findings that the loosely packed precursor RNP undergoes a dramatic conformational change as it compacts into its active form. Our results provide insights into the quaternary arrangement of these RNP complexes in solution, an important step to understanding the transition of the group II intron from the precursor to a species fully active for DNA invasion.

Differential Regulation of Native Estrogen Receptor-Regulatory Elements by Estradiol, Tamoxifen, and Raloxifene

PubMed Central

Levy, Nitzan; Tatomer, Dierdre; Herber, Candice B.; Zhao, Xiaoyue; Tang, Hui; Sargeant, Toby; Ball, Lonnele J.; Summers, Jonathan; Speed, Terence P.; Leitman, Dale C.

2008-01-01

Estrogen receptors (ERs) regulate gene transcription by interacting with regulatory elements. Most information regarding how ER activates genes has come from studies using a small set of target genes or simple consensus sequences such as estrogen response element, activator protein 1, and Sp1 elements. However, these elements cannot explain the differences in gene regulation patterns and clinical effects observed with estradiol (E2) and selective estrogen receptor modulators. To obtain a greater understanding of how E2 and selective estrogen receptor modulators differentially regulate genes, it is necessary to investigate their action on a more comprehensive set of native regulatory elements derived from ER target genes. Here we used chromatin immunoprecipitation-cloning and sequencing to isolate 173 regulatory elements associated with ERα. Most elements were found in the introns (38%) and regions greater than 10 kb upstream of the transcription initiation site (38%); 24% of the elements were found in the proximal promoter region (<10 kb). Only 11% of the elements contained a classical estrogen response element; 23% of the elements did not have any known response elements, including one derived from the naked cuticle homolog gene, which was associated with the recruitment of p160 coactivators. Transfection studies found that 80% of the 173 elements were regulated by E2, raloxifene, or tamoxifen with ERα or ERβ. Tamoxifen was more effective than raloxifene at activating the elements with ERα, whereas raloxifene was superior with ERβ. Our findings demonstrate that E2, tamoxifen, and raloxifene differentially regulate native ER-regulatory elements isolated by chromatin immunoprecipitation with ERα and ERβ. PMID:17962382

Optimization of a novel biophysical model using large scale in vivo antisense hybridization data displays improved prediction capabilities of structurally accessible RNA regions.

PubMed

Vazquez-Anderson, Jorge; Mihailovic, Mia K; Baldridge, Kevin C; Reyes, Kristofer G; Haning, Katie; Cho, Seung Hee; Amador, Paul; Powell, Warren B; Contreras, Lydia M

2017-05-19

Current approaches to design efficient antisense RNAs (asRNAs) rely primarily on a thermodynamic understanding of RNA-RNA interactions. However, these approaches depend on structure predictions and have limited accuracy, arguably due to overlooking important cellular environment factors. In this work, we develop a biophysical model to describe asRNA-RNA hybridization that incorporates in vivo factors using large-scale experimental hybridization data for three model RNAs: a group I intron, CsrB and a tRNA. A unique element of our model is the estimation of the availability of the target region to interact with a given asRNA using a differential entropic consideration of suboptimal structures. We showcase the utility of this model by evaluating its prediction capabilities in four additional RNAs: a group II intron, Spinach II, 2-MS2 binding domain and glgC 5΄ UTR. Additionally, we demonstrate the applicability of this approach to other bacterial species by predicting sRNA-mRNA binding regions in two newly discovered, though uncharacterized, regulatory RNAs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Extended HSR/CARD domain mediates AIRE binding to DNA

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

Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid

Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved inmore » AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.« less

The Arabidopsis homolog of human minor spliceosomal protein U11-48K plays a crucial role in U12 intron splicing and plant development

PubMed Central

Xu, Tao; Kim, Bo Mi; Kwak, Kyung Jin; Jung, Hyun Ju; Kang, Hunseung

2016-01-01

The minor U12 introns are removed from precursor mRNAs by the U12 intron-specific minor spliceosome. Among the seven ribonucleoproteins unique to the minor spliceosome, denoted as U11/U12-20K, U11/U12-25K, U11/U12-31K, U11/U12-65K, U11-35K, U11-48K, and U11-59K, the roles of only U11/U12-31K and U11/U12-65K have been demonstrated in U12 intron splicing and plant development. Here, the functional role of the Arabidopsis homolog of human U11-48K in U12 intron splicing and the development of Arabidopsis thaliana was examined using transgenic knockdown plants. The u11-48k mutants exhibited several defects in growth and development, such as severely arrested primary inflorescence stems, formation of serrated leaves, production of many rosette leaves after bolting, and delayed senescence. The splicing of most U12 introns analyzed was impaired in the u11-48k mutants. Comparative analysis of the splicing defects and phenotypes among the u11/u12-31k, u11-48k, and u11/12-65k mutants showed that the severity of abnormal development was closely correlated with the degree of impairment in U12 intron splicing. Taken together, these results provide compelling evidence that the Arabidopsis homolog of human U11-48K protein, as well as U11/U12-31K and U11/U12-65K proteins, is necessary for correct splicing of U12 introns and normal plant growth and development. PMID:27091878

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

PubMed Central

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-01-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

PubMed

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-12-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

Analysis of the Mitochondrial Genome in Hypomyces aurantius Reveals a Novel Twintron Complex in Fungi.

PubMed

Deng, Youjin; Zhang, Qihui; Ming, Ray; Lin, Longji; Lin, Xiangzhi; Lin, Yiying; Li, Xiao; Xie, Baogui; Wen, Zhiqiang

2016-06-30

Hypomyces aurantius is a mycoparasite that causes cobweb disease, a most serious disease of cultivated mushrooms. Intra-species identification is vital for disease control, however the lack of genomic data makes development of molecular markers challenging. Small size, high copy number, and high mutation rate of fungal mitochondrial genome makes it a good candidate for intra and inter species differentiation. In this study, the mitochondrial genome of H. H.a0001 was determined from genomic DNA using Illumina sequencing. The roughly 72 kb genome shows all major features found in other Hypocreales: 14 common protein genes, large and small subunit rRNAs genes and 27 tRNAs genes. Gene arrangement comparison showed conserved gene orders in Hypocreales mitochondria are relatively conserved, with the exception of Acremonium chrysogenum and Acremonium implicatum. Mitochondrial genome comparison also revealed that intron length primarily contributes to mitogenome size variation. Seventeen introns were detected in six conserved genes: five in cox1, four in rnl, three in cob, two each in atp6 and cox3, and one in cox2. Four introns were found to contain two introns or open reading frames: cox3-i2 is a twintron containing two group IA type introns; cox2-i1 is a group IB intron encoding two homing endonucleases; and cox1-i4 and cox1-i3 both contain two open reading frame (ORFs). Analyses combining secondary intronic structures, insertion sites, and similarities of homing endonuclease genes reveal two group IA introns arranged side by side within cox3-i2. Mitochondrial data for H. aurantius provides the basis for further studies relating to population genetics and species identification.

Analysis of the Mitochondrial Genome in Hypomyces aurantius Reveals a Novel Twintron Complex in Fungi

PubMed Central

Deng, Youjin; Zhang, Qihui; Ming, Ray; Lin, Longji; Lin, Xiangzhi; Lin, Yiying; Li, Xiao; Xie, Baogui; Wen, Zhiqiang

2016-01-01

Hypomyces aurantius is a mycoparasite that causes cobweb disease, a most serious disease of cultivated mushrooms. Intra-species identification is vital for disease control, however the lack of genomic data makes development of molecular markers challenging. Small size, high copy number, and high mutation rate of fungal mitochondrial genome makes it a good candidate for intra and inter species differentiation. In this study, the mitochondrial genome of H. H.a0001 was determined from genomic DNA using Illumina sequencing. The roughly 72 kb genome shows all major features found in other Hypocreales: 14 common protein genes, large and small subunit rRNAs genes and 27 tRNAs genes. Gene arrangement comparison showed conserved gene orders in Hypocreales mitochondria are relatively conserved, with the exception of Acremonium chrysogenum and Acremonium implicatum. Mitochondrial genome comparison also revealed that intron length primarily contributes to mitogenome size variation. Seventeen introns were detected in six conserved genes: five in cox1, four in rnl, three in cob, two each in atp6 and cox3, and one in cox2. Four introns were found to contain two introns or open reading frames: cox3-i2 is a twintron containing two group IA type introns; cox2-i1 is a group IB intron encoding two homing endonucleases; and cox1-i4 and cox1-i3 both contain two open reading frame (ORFs). Analyses combining secondary intronic structures, insertion sites, and similarities of homing endonuclease genes reveal two group IA introns arranged side by side within cox3-i2. Mitochondrial data for H. aurantius provides the basis for further studies relating to population genetics and species identification. PMID:27376282

Phylogenomic Resolution of the Phylogeny of Laurasiatherian Mammals: Exploring Phylogenetic Signals within Coding and Noncoding Sequences.

PubMed

Chen, Meng-Yun; Liang, Dan; Zhang, Peng

2017-08-01

The interordinal relationships of Laurasiatherian mammals are currently one of the most controversial questions in mammalian phylogenetics. Previous studies mainly relied on coding sequences (CDS) and seldom used noncoding sequences. Here, by data mining public genome data, we compiled an intron data set of 3,638 genes (all introns from a protein-coding gene are considered as a gene) (19,055,073 bp) and a CDS data set of 10,259 genes (20,994,285 bp), covering all major lineages of Laurasiatheria (except Pholidota). We found that the intron data contained stronger and more congruent phylogenetic signals than the CDS data. In agreement with this observation, concatenation and species-tree analyses of the intron data set yielded well-resolved and identical phylogenies, whereas the CDS data set produced weakly supported and incongruent results. Further analyses showed that the phylogeny inferred from the intron data is highly robust to data subsampling and change in outgroup, but the CDS data produced unstable results under the same conditions. Interestingly, gene tree statistical results showed that the most frequently observed gene tree topologies for the CDS and intron data are identical, suggesting that the major phylogenetic signal within the CDS data is actually congruent with that within the intron data. Our final result of Laurasiatheria phylogeny is (Eulipotyphla,((Chiroptera, Perissodactyla),(Carnivora, Cetartiodactyla))), favoring a close relationship between Chiroptera and Perissodactyla. Our study 1) provides a well-supported phylogenetic framework for Laurasiatheria, representing a step towards ending the long-standing "hard" polytomy and 2) argues that intron within genome data is a promising data resource for resolving rapid radiation events across the tree of life. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Frequencies of VNTR and RFLP polymorphisms associated with factor VIII gene in Singapore

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

Fong, I.; Lai, P.S.; Ouah, T.C.

1994-09-01

The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1more » = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.« less

Genome-wide identification, phylogenetic classification, and exon-intron structure characterisation of the tubulin and actin genes in flax (Linum usitatissimum).

PubMed

Pydiura, Nikolay; Pirko, Yaroslav; Galinousky, Dmitry; Postovoitova, Anastasiia; Yemets, Alla; Kilchevsky, Aleksandr; Blume, Yaroslav

2018-06-08

Flax (Linum usitatissimum L.) is a valuable food and fiber crop cultivated for its quality fiber and seed oil. α-, β-, γ-tubulins and actins are the main structural proteins of the cytoskeleton. α- and γ-tubulin and actin genes have not been characterized yet in the flax genome. In this study, we have identified 6 α-tubulin genes, 13 β-tubulin genes, 2 γ-tubulin genes, and 15 actin genes in the flax genome and analysed the phylogenetic relationships between flax and A. thaliana tubulin and actin genes. Six α-tubulin genes are represented by 3 paralogous pairs, among 13 β-tubulin genes 7 different isotypes can be distinguished, 6 of which are encoded by two paralogous genes each. γ-tubulin is represented by a paralogous pair of genes one of which may be not functional. Fifteen actin genes represent 7 paralogous pairs - 7 actin isotypes and a sequentially duplicated copy of one of the genes of one of the isotypes. Exon-intron structure analysis has shown intron length polymorphism within the β-tubulin genes and intron number variation among the α-tubulin gene: 3 or 4 introns are found in two or four genes, respectively. Intron positioning occurs at conservative sites, as observed in numerous other plant species. Flax actin genes show both intron length polymorphisms and variation in the number of intron that may be 2 or 3. These data will be useful to support further studies on the specificity, functioning, regulation and evolution of the flax cytoskeleton proteins. This article is protected by copyright. All rights reserved.

A reconfigurable continuous-flow fluidic routing fabric using a modular, scalable primitive.

PubMed

Silva, Ryan; Bhatia, Swapnil; Densmore, Douglas

2016-07-05

Microfluidic devices, by definition, are required to move liquids from one physical location to another. Given a finite and frequently fixed set of physical channels to route fluids, a primitive design element that allows reconfigurable routing of that fluid from any of n input ports to any n output ports will dramatically change the paradigms by which these chips are designed and applied. Furthermore, if these elements are "regular" regarding their design, the programming and fabrication of these elements becomes scalable. This paper presents such a design element called a transposer. We illustrate the design, fabrication and operation of a single transposer. We then scale this design to create a programmable fabric towards a general-purpose, reconfigurable microfluidic platform analogous to the Field Programmable Gate Array (FPGA) found in digital electronics.

Surface Buildup Scenarios and Outpost Architectures for Lunar Exploration

NASA Technical Reports Server (NTRS)

Mazanek, Daniel D.; Troutman, Patrick A.; Culbert, Christopher J.; Leonard, Matthew J.; Spexarth, Gary R.

2009-01-01

The Constellation Program Architecture Team and the Lunar Surface Systems Project Office have developed an initial set of lunar surface buildup scenarios and associated polar outpost architectures, along with preliminary supporting element and system designs in support of NASA's Exploration Strategy. The surface scenarios are structured in such a way that outpost assembly can be suspended at any time to accommodate delivery contingencies or changes in mission emphasis. The modular nature of the architectures mitigates the impact of the loss of any one element and enhances the ability of international and commercial partners to contribute elements and systems. Additionally, the core lunar surface system technologies and outpost operations concepts are applicable to future Mars exploration. These buildup scenarios provide a point of departure for future trades and assessments of alternative architectures and surface elements.

Development of single-copy nuclear intron markers for species-level phylogenetics: Case study with Paullinieae (Sapindaceae).

PubMed

Chery, Joyce G; Sass, Chodon; Specht, Chelsea D

2017-09-01

We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline. Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a "pseudoreference" closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae. This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo.

Tobacco chloroplast tRNALys(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron

PubMed Central

Sugita, Mamoru; Shinozaki, Kazuo; Sugiura, Masahiro

1985-01-01

The nucleotide sequence of a tRNALys(UUU) gene on tobacco (Nicotiana tabacum) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNAGly(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long. Images PMID:16593561

Tobacco chloroplast tRNA(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron.

PubMed

Sugita, M; Shinozaki, K; Sugiura, M

1985-06-01

The nucleotide sequence of a tRNA(Lys)(UUU) gene on tobacco (Nicotiana tabacum) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNA(Gly)(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long.

Separate cortical networks involved in music perception: preliminary functional MRI evidence for modularity of music processing.

PubMed

Schmithorst, Vincent J

2005-04-01

Music perception is a quite complex cognitive task, involving the perception and integration of various elements including melody, harmony, pitch, rhythm, and timbre. A preliminary functional MRI investigation of music perception was performed, using a simplified passive listening task. Group independent component analysis (ICA) was used to separate out various components involved in music processing, as the hemodynamic responses are not known a priori. Various components consistent with auditory processing, expressive language, syntactic processing, and visual association were found. The results are discussed in light of various hypotheses regarding modularity of music processing and its overlap with language processing. The results suggest that, while some networks overlap with ones used for language processing, music processing may involve its own domain-specific processing subsystems.

Image Navigation and Registration Performance Assessment Evaluation Tools for GOES-R ABI and GLM

NASA Technical Reports Server (NTRS)

Houchin, Scott; Porter, Brian; Graybill, Justin; Slingerland, Philip

2017-01-01

The GOES-R Flight Project has developed an Image Navigation and Registration (INR) Performance Assessment Tool Set (IPATS) for measuring Advanced Baseline Imager (ABI) and Geostationary Lightning Mapper (GLM) INR performance metrics in the post-launch period for performance evaluation and long term monitoring. IPATS utilizes a modular algorithmic design to allow user selection of data processing sequences optimized for generation of each INR metric. This novel modular approach minimizes duplication of common processing elements, thereby maximizing code efficiency and speed. Fast processing is essential given the large number of sub-image registrations required to generate INR metrics for the many images produced over a 24 hour evaluation period. This paper describes the software design and implementation of IPATS and provides preliminary test results.

Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing

PubMed Central

Kwak, Hojoong; Fuda, Nicholas J.; Core, Leighton J.; Lis, John T.

2014-01-01

Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter. PMID:23430654

Thermodynamic Modeling of Donor Splice Site Recognition in pre-mRNA

NASA Astrophysics Data System (ADS)

Aalberts, Daniel P.; Garland, Jeffrey A.

2004-03-01

When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 snRNA with the donor (5') splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our Finding with Binding method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

Thermodynamic modeling of donor splice site recognition in pre-mRNA

NASA Astrophysics Data System (ADS)

Garland, Jeffrey A.; Aalberts, Daniel P.

2004-04-01

When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 small nuclear RNA with the donor ( 5' ) splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our “finding with binding” method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

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

PubMed Central

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

2016-01-01

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

Molecular Evolution of the Metallothionein Gene Mtn in the Melanogaster Species Group: Results from Drosophila Ananassae

PubMed Central

Stephan, W.; Rodriguez, V. S.; Zhou, B.; Parsch, J.

1994-01-01

Three distinctly different alleles of the metallothionein gene Mtn have been identified in natural Drosophila melanogaster populations: Mtn(.3), Mtn(1), and Dp(Mtn(1)), where the latter designates a tandem duplication of Mtn(1). In Drosophila simulans, only Mtn(.3)-type alleles have been found. It has been suggested that Mtn(.3) is the ancestral allele and demonstrated that a presumed two-step transition from Mtn(.3) to Mtn(1) to Dp(Mtn(1)) is accompanied by an approximate 5-fold increase in RNA levels. We analyzed the evolutionary genetics of the Mtn locus of Drosophila ananassae, a distant relative of D. melanogaster and D. simulans within the melanogaster species group. The Mtn gene of D. ananassae is most similar to Mtn(.3). (i) it is identical with Mtn(.3) at the amino acid level, but differs from Mtn(1) in its terminal codon; (ii) its 3' UTR contains a characteristic extra DNA segment of about 50 bp which is present in Mtn(.3), but lacking in Mtn(1); (iii) duplications of Mtn were not found in a worldwide sample of 110 wild D. ananassae chromosomes. However, the intron of the Mtn gene in D. ananassae is only 69 bp long, whereas the length of the Mtn(.3) and Mtn(1) introns is 265 bp; and it lacks a polypyrimidine stretch upstream of the 3' splice site in contrast to the much greater pyrimidine-richness found in the Mtn(.3) and Mtn(1) introns. A short intron (67 bp) was also identified in a D. pseudoobscura Mtn allele, suggesting that the short intron is the ancestral form and that the transition from the short to the long intron occurred within the melanogaster species group. We discuss the significance of this observation with regard to the recently proposed classification of D. melanogaster introns into two groups: short introns (<90 bp) which tend to lack polypyrimidine stretches, and longer ones which have strong 3' splice signals similar to mammalian introns. A database search revealed that this length dimorphism is an evolutionarily conserved feature of Drosophila introns; transitions from one size class to the other appear to be rare between closely related species (e.g., within the melanogaster subgroup). PMID:8001781

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

PubMed

Leung, Wilson; Shaffer, Christopher D; Reed, Laura K; Smith, Sheryl T; Barshop, William; Dirkes, William; Dothager, Matthew; Lee, Paul; Wong, Jeannette; Xiong, David; Yuan, Han; Bedard, James E J; Machone, Joshua F; Patterson, Seantay D; Price, Amber L; Turner, Bryce A; Robic, Srebrenka; Luippold, Erin K; McCartha, Shannon R; Walji, Tezin A; Walker, Chelsea A; Saville, Kenneth; Abrams, Marita K; Armstrong, Andrew R; Armstrong, William; Bailey, Robert J; Barberi, Chelsea R; Beck, Lauren R; Blaker, Amanda L; Blunden, Christopher E; Brand, Jordan P; Brock, Ethan J; Brooks, Dana W; Brown, Marie; Butzler, Sarah C; Clark, Eric M; Clark, Nicole B; Collins, Ashley A; Cotteleer, Rebecca J; Cullimore, Peterson R; Dawson, Seth G; Docking, Carter T; Dorsett, Sasha L; Dougherty, Grace A; Downey, Kaitlyn A; Drake, Andrew P; Earl, Erica K; Floyd, Trevor G; Forsyth, Joshua D; Foust, Jonathan D; Franchi, Spencer L; Geary, James F; Hanson, Cynthia K; Harding, Taylor S; Harris, Cameron B; Heckman, Jonathan M; Holderness, Heather L; Howey, Nicole A; Jacobs, Dontae A; Jewell, Elizabeth S; Kaisler, Maria; Karaska, Elizabeth A; Kehoe, James L; Koaches, Hannah C; Koehler, Jessica; Koenig, Dana; Kujawski, Alexander J; Kus, Jordan E; Lammers, Jennifer A; Leads, Rachel R; Leatherman, Emily C; Lippert, Rachel N; Messenger, Gregory S; Morrow, Adam T; Newcomb, Victoria; Plasman, Haley J; Potocny, Stephanie J; Powers, Michelle K; Reem, Rachel M; Rennhack, Jonathan P; Reynolds, Katherine R; Reynolds, Lyndsey A; Rhee, Dong K; Rivard, Allyson B; Ronk, Adam J; Rooney, Meghan B; Rubin, Lainey S; Salbert, Luke R; Saluja, Rasleen K; Schauder, Taylor; Schneiter, Allison R; Schulz, Robert W; Smith, Karl E; Spencer, Sarah; Swanson, Bryant R; Tache, Melissa A; Tewilliager, Ashley A; Tilot, Amanda K; VanEck, Eve; Villerot, Matthew M; Vylonis, Megan B; Watson, David T; Wurzler, Juliana A; Wysocki, Lauren M; Yalamanchili, Monica; Zaborowicz, Matthew A; Emerson, Julia A; Ortiz, Carlos; Deuschle, Frederic J; DiLorenzo, Lauren A; Goeller, Katie L; Macchi, Christopher R; Muller, Sarah E; Pasierb, Brittany D; Sable, Joseph E; Tucci, Jessica M; Tynon, Marykathryn; Dunbar, David A; Beken, Levent H; Conturso, Alaina C; Danner, Benjamin L; DeMichele, Gabriella A; Gonzales, Justin A; Hammond, Maureen S; Kelley, Colleen V; Kelly, Elisabeth A; Kulich, Danielle; Mageeney, Catherine M; McCabe, Nikie L; Newman, Alyssa M; Spaeder, Lindsay A; Tumminello, Richard A; Revie, Dennis; Benson, Jonathon M; Cristostomo, Michael C; DaSilva, Paolo A; Harker, Katherine S; Jarrell, Jenifer N; Jimenez, Luis A; Katz, Brandon M; Kennedy, William R; Kolibas, Kimberly S; LeBlanc, Mark T; Nguyen, Trung T; Nicolas, Daniel S; Patao, Melissa D; Patao, Shane M; Rupley, Bryan J; Sessions, Bridget J; Weaver, Jennifer A; Goodman, Anya L; Alvendia, Erica L; Baldassari, Shana M; Brown, Ashley S; Chase, Ian O; Chen, Maida; Chiang, Scott; Cromwell, Avery B; Custer, Ashley F; DiTommaso, Tia M; El-Adaimi, Jad; Goscinski, Nora C; Grove, Ryan A; Gutierrez, Nestor; Harnoto, Raechel S; Hedeen, Heather; Hong, Emily L; Hopkins, Barbara L; Huerta, Vilma F; Khoshabian, Colin; LaForge, Kristin M; Lee, Cassidy T; Lewis, Benjamin M; Lydon, Anniken M; Maniaci, Brian J; Mitchell, Ryan D; Morlock, Elaine V; Morris, William M; Naik, Priyanka; Olson, Nicole C; Osterloh, Jeannette M; Perez, Marcos A; Presley, Jonathan D; Randazzo, Matt J; Regan, Melanie K; Rossi, Franca G; Smith, Melanie A; Soliterman, Eugenia A; Sparks, Ciani J; Tran, Danny L; Wan, Tiffany; Welker, Anne A; Wong, Jeremy N; Sreenivasan, Aparna; Youngblom, Jim; Adams, Andrew; Alldredge, Justin; Bryant, Ashley; Carranza, David; Cifelli, Alyssa; Coulson, Kevin; Debow, Calise; Delacruz, Noelle; Emerson, Charlene; Farrar, Cassandra; Foret, Don; Garibay, Edgar; Gooch, John; Heslop, Michelle; Kaur, Sukhjit; Khan, Ambreen; Kim, Van; Lamb, Travis; Lindbeck, Peter; Lucas, Gabi; Macias, Elizabeth; Martiniuc, Daniela; Mayorga, Lissett; Medina, Joseph; Membreno, Nelson; Messiah, Shady; Neufeld, Lacey; Nguyen, San Francisco; Nichols, Zachary; Odisho, George; Peterson, Daymon; Rodela, Laura; Rodriguez, Priscilla; Rodriguez, Vanessa; Ruiz, Jorge; Sherrill, Will; Silva, Valeria; Sparks, Jeri; Statton, Geeta; Townsend, Ashley; Valdez, Isabel; Waters, Mary; Westphal, Kyle; Winkler, Stacey; Zumkehr, Joannee; DeJong, Randall J; Hoogewerf, Arlene J; Ackerman, Cheri M; Armistead, Isaac O; Baatenburg, Lara; Borr, Matthew J; Brouwer, Lindsay K; Burkhart, Brandon J; Bushhouse, Kelsey T; Cesko, Lejla; Choi, Tiffany Y Y; Cohen, Heather; Damsteegt, Amanda M; Darusz, Jess M; Dauphin, Cory M; Davis, Yelena P; Diekema, Emily J; Drewry, Melissa; Eisen, Michelle E M; Faber, Hayley M; Faber, Katherine J; Feenstra, Elizabeth; Felzer-Kim, Isabella T; Hammond, Brandy L; Hendriksma, Jesse; Herrold, Milton R; Hilbrands, Julia A; Howell, Emily J; Jelgerhuis, Sarah A; Jelsema, Timothy R; Johnson, Benjamin K; Jones, Kelly K; Kim, Anna; Kooienga, Ross D; Menyes, Erika E; Nollet, Eric A; Plescher, Brittany E; Rios, Lindsay; Rose, Jenny L; Schepers, Allison J; Scott, Geoff; Smith, Joshua R; Sterling, Allison M; Tenney, Jenna C; Uitvlugt, Chris; VanDyken, Rachel E; VanderVennen, Marielle; Vue, Samantha; Kokan, Nighat P; Agbley, Kwabea; Boham, Sampson K; Broomfield, Daniel; Chapman, Kayla; Dobbe, Ali; Dobbe, Ian; Harrington, William; Ibrahem, Marwan; Kennedy, Andre; Koplinsky, Chad A; Kubricky, Cassandra; Ladzekpo, Danielle; Pattison, Claire; Ramirez, Roman E; Wande, Lucia; Woehlke, Sarah; Wawersik, Matthew; Kiernan, Elizabeth; Thompson, Jeffrey S; Banker, Roxanne; Bartling, Justina R; Bhatiya, Chinmoy I; Boudoures, Anna L; Christiansen, Lena; Fosselman, Daniel S; French, Kristin M; Gill, Ishwar S; Havill, Jessen T; Johnson, Jaelyn L; Keny, Lauren J; Kerber, John M; Klett, Bethany M; Kufel, Christina N; May, Francis J; Mecoli, Jonathan P; Merry, Callie R; Meyer, Lauren R; Miller, Emily G; Mullen, Gregory J; Palozola, Katherine C; Pfeil, Jacob J; Thomas, Jessica G; Verbofsky, Evan M; Spana, Eric P; Agarwalla, Anant; Chapman, Julia; Chlebina, Ben; Chong, Insun; Falk, I N; Fitzgibbons, John D; Friedman, Harrison; Ighile, Osagie; Kim, Andrew J; Knouse, Kristin A; Kung, Faith; Mammo, Danny; Ng, Chun Leung; Nikam, Vinayak S; Norton, Diana; Pham, Philip; Polk, Jessica W; Prasad, Shreya; Rankin, Helen; Ratliff, Camille D; Scala, Victoria; Schwartz, Nicholas U; Shuen, Jessica A; Xu, Amy; Xu, Thomas Q; Zhang, Yi; Rosenwald, Anne G; Burg, Martin G; Adams, Stephanie J; Baker, Morgan; Botsford, Bobbi; Brinkley, Briana; Brown, Carter; Emiah, Shadie; Enoch, Erica; Gier, Chad; Greenwell, Alyson; Hoogenboom, Lindsay; Matthews, Jordan E; McDonald, Mitchell; Mercer, Amanda; Monsma, Nicholaus; Ostby, Kristine; Ramic, Alen; Shallman, Devon; Simon, Matthew; Spencer, Eric; Tomkins, Trisha; Wendland, Pete; Wylie, Anna; Wolyniak, Michael J; Robertson, Gregory M; Smith, Samuel I; DiAngelo, Justin R; Sassu, Eric D; Bhalla, Satish C; Sharif, Karim A; Choeying, Tenzin; Macias, Jason S; Sanusi, Fareed; Torchon, Karvyn; Bednarski, April E; Alvarez, Consuelo J; Davis, Kristen C; Dunham, Carrie A; Grantham, Alaina J; Hare, Amber N; Schottler, Jennifer; Scott, Zackary W; Kuleck, Gary A; Yu, Nicole S; Kaehler, Marian M; Jipp, Jacob; Overvoorde, Paul J; Shoop, Elizabeth; Cyrankowski, Olivia; Hoover, Betsy; Kusner, Matt; Lin, Devry; Martinov, Tijana; Misch, Jonathan; Salzman, Garrett; Schiedermayer, Holly; Snavely, Michael; Zarrasola, Stephanie; Parrish, Susan; Baker, Atlee; Beckett, Alissa; Belella, Carissa; Bryant, Julie; Conrad, Turner; Fearnow, Adam; Gomez, Carolina; Herbstsomer, Robert A; Hirsch, Sarah; Johnson, Christen; Jones, Melissa; Kabaso, Rita; Lemmon, Eric; Vieira, Carolina Marques Dos Santos; McFarland, Darryl; McLaughlin, Christopher; Morgan, Abbie; Musokotwane, Sepo; Neutzling, William; Nietmann, Jana; Paluskievicz, Christina; Penn, Jessica; Peoples, Emily; Pozmanter, Caitlin; Reed, Emily; Rigby, Nichole; Schmidt, Lasse; Shelton, Micah; Shuford, Rebecca; Tirasawasdichai, Tiara; Undem, Blair; Urick, Damian; Vondy, Kayla; Yarrington, Bryan; Eckdahl, Todd T; Poet, Jeffrey L; Allen, Alica B; Anderson, John E; Barnett, Jason M; Baumgardner, Jordan S; Brown, Adam D; Carney, Jordan E; Chavez, Ramiro A; Christgen, Shelbi L; Christie, Jordan S; Clary, Andrea N; Conn, Michel A; Cooper, Kristen M; Crowley, Matt J; Crowley, Samuel T; Doty, Jennifer S; Dow, Brian A; Edwards, Curtis R; Elder, Darcie D; Fanning, John P; Janssen, Bridget M; Lambright, Anthony K; Lane, Curtiss E; Limle, Austin B; Mazur, Tammy; McCracken, Marly R; McDonough, Alexa M; Melton, Amy D; Minnick, Phillip J; Musick, Adam E; Newhart, William H; Noynaert, Joseph W; Ogden, Bradley J; Sandusky, Michael W; Schmuecker, Samantha M; Shipman, Anna L; Smith, Anna L; Thomsen, Kristen M; Unzicker, Matthew R; Vernon, William B; Winn, Wesley W; Woyski, Dustin S; Zhu, Xiao; Du, Chunguang; Ament, Caitlin; Aso, Soham; Bisogno, Laura Simone; Caronna, Jason; Fefelova, Nadezhda; Lopez, Lenin; Malkowitz, Lorraine; Marra, Jonathan; Menillo, Daniella; Obiorah, Ifeanyi; Onsarigo, Eric Nyabeta; Primus, Shekerah; Soos, Mahdi; Tare, Archana; Zidan, Ameer; Jones, Christopher J; Aronhalt, Todd; Bellush, James M; Burke, Christa; DeFazio, Steve; Does, Benjamin R; Johnson, Todd D; Keysock, Nicholas; Knudsen, Nelson H; Messler, James; Myirski, Kevin; Rekai, Jade Lea; Rempe, Ryan Michael; Salgado, Michael S; Stagaard, Erica; Starcher, Justin R; Waggoner, Andrew W; Yemelyanova, Anastasia K; Hark, Amy T; Bertolet, Anne; Kuschner, Cyrus E; Parry, Kesley; Quach, Michael; Shantzer, Lindsey; Shaw, Mary E; Smith, Mary A; Glenn, Omolara; Mason, Portia; Williams, Charlotte; Key, S Catherine Silver; Henry, Tyneshia C P; Johnson, Ashlee G; White, Jackie X; Haberman, Adam; Asinof, Sam; Drumm, Kelly; Freeburg, Trip; Safa, Nadia; Schultz, Darrin; Shevin, Yakov; Svoronos, Petros; Vuong, Tam; Wellinghoff, Jules; Hoopes, Laura L M; Chau, Kim M; Ward, Alyssa; Regisford, E Gloria C; Augustine, LaJerald; Davis-Reyes, Brionna; Echendu, Vivienne; Hales, Jasmine; Ibarra, Sharon; Johnson, Lauriaun; Ovu, Steven; Braverman, John M; Bahr, Thomas J; Caesar, Nicole M; Campana, Christopher; Cassidy, Daniel W; Cognetti, Peter A; English, Johnathan D; Fadus, Matthew C; Fick, Cameron N; Freda, Philip J; Hennessy, Bryan M; Hockenberger, Kelsey; Jones, Jennifer K; King, Jessica E; Knob, Christopher R; Kraftmann, Karen J; Li, Linghui; Lupey, Lena N; Minniti, Carl J; Minton, Thomas F; Moran, Joseph V; Mudumbi, Krishna; Nordman, Elizabeth C; Puetz, William J; Robinson, Lauren M; Rose, Thomas J; Sweeney, Edward P; Timko, Ashley S; Paetkau, Don W; Eisler, Heather L; Aldrup, Megan E; Bodenberg, Jessica M; Cole, Mara G; Deranek, Kelly M; DeShetler, Megan; Dowd, Rose M; Eckardt, Alexandra K; Ehret, Sharon C; Fese, Jessica; Garrett, Amanda D; Kammrath, Anna; Kappes, Michelle L; Light, Morgan R; Meier, Anne C; O'Rouke, Allison; Perella, Mallory; Ramsey, Kimberley; Ramthun, Jennifer R; Reilly, Mary T; Robinett, Deirdre; Rossi, Nadine L; Schueler, Mary Grace; Shoemaker, Emma; Starkey, Kristin M; Vetor, Ashley; Vrable, Abby; Chandrasekaran, Vidya; Beck, Christopher; Hatfield, Kristen R; Herrick, Douglas A; Khoury, Christopher B; Lea, Charlotte; Louie, Christopher A; Lowell, Shannon M; Reynolds, Thomas J; Schibler, Jeanine; Scoma, Alexandra H; Smith-Gee, Maxwell T; Tuberty, Sarah; Smith, Christopher D; Lopilato, Jane E; Hauke, Jeanette; Roecklein-Canfield, Jennifer A; Corrielus, Maureen; Gilman, Hannah; Intriago, Stephanie; Maffa, Amanda; Rauf, Sabya A; Thistle, Katrina; Trieu, Melissa; Winters, Jenifer; Yang, Bib; Hauser, Charles R; Abusheikh, Tariq; Ashrawi, Yara; Benitez, Pedro; Boudreaux, Lauren R; Bourland, Megan; Chavez, Miranda; Cruz, Samantha; Elliott, GiNell; Farek, Jesse R; Flohr, Sarah; Flores, Amanda H; Friedrichs, Chelsey; Fusco, Zach; Goodwin, Zane; Helmreich, Eric; Kiley, John; Knepper, John Mark; Langner, Christine; Martinez, Megan; Mendoza, Carlos; Naik, Monal; Ochoa, Andrea; Ragland, Nicolas; Raimey, England; Rathore, Sunil; Reza, Evangelina; Sadovsky, Griffin; Seydoux, Marie-Isabelle B; Smith, Jonathan E; Unruh, Anna K; Velasquez, Vicente; Wolski, Matthew W; Gosser, Yuying; Govind, Shubha; Clarke-Medley, Nicole; Guadron, Leslie; Lau, Dawn; Lu, Alvin; Mazzeo, Cheryl; Meghdari, Mariam; Ng, Simon; Pamnani, Brad; Plante, Olivia; Shum, Yuki Kwan Wa; Song, Roy; Johnson, Diana E; Abdelnabi, Mai; Archambault, Alexi; Chamma, Norma; Gaur, Shailly; Hammett, Deborah; Kandahari, Adrese; Khayrullina, Guzal; Kumar, Sonali; Lawrence, Samantha; Madden, Nigel; Mandelbaum, Max; Milnthorp, Heather; Mohini, Shiv; Patel, Roshni; Peacock, Sarah J; Perling, Emily; Quintana, Amber; Rahimi, Michael; Ramirez, Kristen; Singhal, Rishi; Weeks, Corinne; Wong, Tiffany; Gillis, Aubree T; Moore, Zachary D; Savell, Christopher D; Watson, Reece; Mel, Stephanie F; Anilkumar, Arjun A; Bilinski, Paul; Castillo, Rostislav; Closser, Michael; Cruz, Nathalia M; Dai, Tiffany; Garbagnati, Giancarlo F; Horton, Lanor S; Kim, Dongyeon; Lau, Joyce H; Liu, James Z; Mach, Sandy D; Phan, Thu A; Ren, Yi; Stapleton, Kenneth E; Strelitz, Jean M; Sunjed, Ray; Stamm, Joyce; Anderson, Morgan C; Bonifield, Bethany Grace; Coomes, Daniel; Dillman, Adam; Durchholz, Elaine J; Fafara-Thompson, Antoinette E; Gross, Meleah J; Gygi, Amber M; Jackson, Lesley E; Johnson, Amy; Kocsisova, Zuzana; Manghelli, Joshua L; McNeil, Kylie; Murillo, Michael; Naylor, Kierstin L; Neely, Jessica; Ogawa, Emmy E; Rich, Ashley; Rogers, Anna; Spencer, J Devin; Stemler, Kristina M; Throm, Allison A; Van Camp, Matt; Weihbrecht, Katie; Wiles, T Aaron; Williams, Mallory A; Williams, Matthew; Zoll, Kyle; Bailey, Cheryl; Zhou, Leming; Balthaser, Darla M; Bashiri, Azita; Bower, Mindy E; Florian, Kayla A; Ghavam, Nazanin; Greiner-Sosanko, Elizabeth S; Karim, Helmet; Mullen, Victor W; Pelchen, Carly E; Yenerall, Paul M; Zhang, Jiayu; Rubin, Michael R; Arias-Mejias, Suzette M; Bermudez-Capo, Armando G; Bernal-Vega, Gabriela V; Colon-Vazquez, Mariela; Flores-Vazquez, Arelys; Gines-Rosario, Mariela; Llavona-Cartagena, Ivan G; Martinez-Rodriguez, Javier O; Ortiz-Fuentes, Lionel; Perez-Colomba, Eliezer O; Perez-Otero, Joseph; Rivera, Elisandra; Rodriguez-Giron, Luke J; Santiago-Sanabria, Arnaldo J; Senquiz-Gonzalez, Andrea M; delValle, Frank R Soto; Vargas-Franco, Dorianmarie; Velázquez-Soto, Karla I; Zambrana-Burgos, Joan D; Martinez-Cruzado, Juan Carlos; Asencio-Zayas, Lillyann; Babilonia-Figueroa, Kevin; Beauchamp-Pérez, Francis D; Belén-Rodríguez, Juliana; Bracero-Quiñones, Luciann; Burgos-Bula, Andrea P; Collado-Méndez, Xavier A; Colón-Cruz, Luis R; Correa-Muller, Ana I; Crooke-Rosado, Jonathan L; Cruz-García, José M; Defendini-Ávila, Marianna; Delgado-Peraza, Francheska M; Feliciano-Cancela, Alex J; Gónzalez-Pérez, Valerie M; Guiblet, Wilfried; Heredia-Negrón, Aldo; Hernández-Muñiz, Jennifer; Irizarry-González, Lourdes N; Laboy-Corales, Ángel L; Llaurador-Caraballo, Gabriela A; Marín-Maldonado, Frances; Marrero-Llerena, Ulises; Martell-Martínez, Héctor A; Martínez-Traverso, Idaliz M; Medina-Ortega, Kiara N; Méndez-Castellanos, Sonya G; Menéndez-Serrano, Krizia C; Morales-Caraballo, Carol I; Ortiz-DeChoudens, Saryleine; Ortiz-Ortiz, Patricia; Pagán-Torres, Hendrick; Pérez-Afanador, Diana; Quintana-Torres, Enid M; Ramírez-Aponte, Edwin G; Riascos-Cuero, Carolina; Rivera-Llovet, Michelle S; Rivera-Pagán, Ingrid T; Rivera-Vicéns, Ramón E; Robles-Juarbe, Fabiola; Rodríguez-Bonilla, Lorraine; Rodríguez-Echevarría, Brian O; Rodríguez-García, Priscila M; Rodríguez-Laboy, Abneris E; Rodríguez-Santiago, Susana; Rojas-Vargas, Michael L; Rubio-Marrero, Eva N; Santiago-Colón, Albeliz; Santiago-Ortiz, Jorge L; Santos-Ramos, Carlos E; Serrano-González, Joseline; Tamayo-Figueroa, Alina M; Tascón-Peñaranda, Edna P; Torres-Castillo, José L; Valentín-Feliciano, Nelson A; Valentín-Feliciano, Yashira M; Vargas-Barreto, Nadyan M; Vélez-Vázquez, Miguel; Vilanova-Vélez, Luis R; Zambrana-Echevarría, Cristina; MacKinnon, Christy; Chung, Hui-Min; Kay, Chris; Pinto, Anthony; Kopp, Olga R; Burkhardt, Joshua; Harward, Chris; Allen, Robert; Bhat, Pavan; Chang, Jimmy Hsiang-Chun; Chen, York; Chesley, Christopher; Cohn, Dara; DuPuis, David; Fasano, Michael; Fazzio, Nicholas; Gavinski, Katherine; Gebreyesus, Heran; Giarla, Thomas; Gostelow, Marcus; Greenstein, Rachel; Gunasinghe, Hashini; Hanson, Casey; Hay, Amanda; He, Tao Jian; Homa, Katie; Howe, Ruth; Howenstein, Jeff; Huang, Henry; Khatri, Aaditya; Kim, Young Lu; Knowles, Olivia; Kong, Sarah; Krock, Rebecca; Kroll, Matt; Kuhn, Julia; Kwong, Matthew; Lee, Brandon; Lee, Ryan; Levine, Kevin; Li, Yedda; Liu, Bo; Liu, Lucy; Liu, Max; Lousararian, Adam; Ma, Jimmy; Mallya, Allyson; Manchee, Charlie; Marcus, Joseph; McDaniel, Stephen; Miller, Michelle L; Molleston, Jerome M; Diez, Cristina Montero; Ng, Patrick; Ngai, Natalie; Nguyen, Hien; Nylander, Andrew; Pollack, Jason; Rastogi, Suchita; Reddy, Himabindu; Regenold, Nathaniel; Sarezky, Jon; Schultz, Michael; Shim, Jien; Skorupa, Tara; Smith, Kenneth; Spencer, Sarah J; Srikanth, Priya; Stancu, Gabriel; Stein, Andrew P; Strother, Marshall; Sudmeier, Lisa; Sun, Mengyang; Sundaram, Varun; Tazudeen, Noor; Tseng, Alan; Tzeng, Albert; Venkat, Rohit; Venkataram, Sandeep; Waldman, Leah; Wang, Tracy; Yang, Hao; Yu, Jack Y; Zheng, Yin; Preuss, Mary L; Garcia, Angelica; Juergens, Matt; Morris, Robert W; Nagengast, Alexis A; Azarewicz, Julie; Carr, Thomas J; Chichearo, Nicole; Colgan, Mike; Donegan, Megan; Gardner, Bob; Kolba, Nik; Krumm, Janice L; Lytle, Stacey; MacMillian, Laurell; Miller, Mary; Montgomery, Andrew; Moretti, Alysha; Offenbacker, Brittney; Polen, Mike; Toth, John; Woytanowski, John; Kadlec, Lisa; Crawford, Justin; Spratt, Mary L; Adams, Ashley L; Barnard, Brianna K; Cheramie, Martin N; Eime, Anne M; Golden, Kathryn L; Hawkins, Allyson P; Hill, Jessica E; Kampmeier, Jessica A; Kern, Cody D; Magnuson, Emily E; Miller, Ashley R; Morrow, Cody M; Peairs, Julia C; Pickett, Gentry L; Popelka, Sarah A; Scott, Alexis J; Teepe, Emily J; TerMeer, Katie A; Watchinski, Carmen A; Watson, Lucas A; Weber, Rachel E; Woodard, Kate A; Barnard, Daron C; Appiah, Isaac; Giddens, Michelle M; McNeil, Gerard P; Adebayo, Adeola; Bagaeva, Kate; Chinwong, Justina; Dol, Chrystel; George, Eunice; Haltaufderhyde, Kirk; Haye, Joanna; Kaur, Manpreet; Semon, Max; Serjanov, Dmitri; Toorie, Anika; Wilson, Christopher; Riddle, Nicole C; Buhler, Jeremy; Mardis, Elaine R; Elgin, Sarah C R

2015-03-04

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu. Copyright © 2015 Leung et al.

Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging

PubMed Central

Pocock, Ginger M.; Zimdars, Laraine L.; Yuan, Ming; Eliceiri, Kevin W.; Ahlquist, Paul; Sherer, Nathan M.

2017-01-01

Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include “burst” RNA nuclear export dynamics regulated by HIV-1’s Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element–specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation. PMID:27903772

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

PubMed

Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

2008-08-22

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

PubMed

Voorbij, Annemarie M W Y; van Steenbeek, Frank G; Vos-Loohuis, Manon; Martens, Ellen E C P; Hanson-Nilsson, Jeanette M; van Oost, Bernard A; Kooistra, Hans S; Leegwater, Peter A

2011-01-01

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs

PubMed Central

Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.

2011-01-01

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism. PMID:22132174

Development of Pulsed Processes for the Manufacture of Solar Cells

NASA Technical Reports Server (NTRS)

1979-01-01

The development status of the process based upon ion implantation for the introduction of junctions and back surface fields is described. A process sequence is presented employing ion implantation and pulse processing. Efforts to improve throughout and descrease process element costs for furnace annealing are described. Design studies for a modular 3,000 wafer per hour pulse processor are discussed.

High School English IV: Comparative Mythology. Revised Edition.

ERIC Educational Resources Information Center

Clark, Karen J.

This modular curriculum for independent study by high school students is a one-fourth unit credit course on Comparative Mythology. The aims of the course are to help the student to: (1) become aware of the orally transmitted culture in their lives; (2) become aware of the mythical element in all of man's history; (3) know the meanings of the…

Psychological Operations: Fighting the War of Ideas

DTIC Science & Technology

2007-05-18

is the success of the Joint Interagency Task Force on the Former Regime Elements (JIATF- FRE) operation to capture Fadhil Ibrahim Habib al-Mashadani... DAPS ), Fly Away Broadcast System (FABS), and Target Audience Analysis Detachment (TAAD). This provides the Brigade a radio development and broadcast...level. Production and dissemination assets must include a Modular Print System (MPS), Deployable Audio Production Suite ( DAPS ), and Special

Multipurpose high-pressure high-temperature diamond-anvil cell with a novel high-precision guiding system and a dual-mode pressurization device

NASA Astrophysics Data System (ADS)

Pippinger, Thomas; Miletich, Ronald; Burchard, Michael

2011-09-01

A novel diamond-anvil cell (DAC) design has been constructed and tested for in situ applications at high-pressure (HP) operations and has proved to be suitable even for HP sample environments at non-ambient temperature conditions. The innovative high-precision guiding mechanism, comparable to a dog clutch, consists of perpendicular planar sliding-plane elements and is integrated directly into the base body of the cylindrically shaped DAC. The combination of two force-generating devices, i.e., mechanical screws and an inflatable gas membrane, allows the user to choose independently between, and to apply individually, two different forcing mechanisms for pressure generation. Both mechanisms are basically independent of each other, but can also be operated simultaneously. The modularity of the DAC design allows for an easy exchange of functional core-element groups optimized not only for various analytical in situ methods but also for HP operation with or without high-temperature (HT) application. For HP-HT experiments a liquid cooling circuit inside the specific inner modular groups has been implemented to obtain a controlled and limited heat distribution within the outer DAC body.

Boiler using combustible fluid

DOEpatents

Baumgartner, H.; Meier, J.G.

1974-07-03

A fluid fuel boiler is described comprising a combustion chamber, a cover on the combustion chamber having an opening for introducing a combustion-supporting gaseous fluid through said openings, means to impart rotation to the gaseous fluid about an axis of the combustion chamber, a burner for introducing a fluid fuel into the chamber mixed with the gaseous fluid for combustion thereof, the cover having a generally frustro-conical configuration diverging from the opening toward the interior of the chamber at an angle of between 15/sup 0/ and 55/sup 0/; means defining said combustion chamber having means defining a plurality of axial hot gas flow paths from a downstream portion of the combustion chamber to flow hot gases into an upstream portion of the combustion chamber, and means for diverting some of the hot gas flow along paths in a direction circumferentially of the combustion chamber, with the latter paths being immersed in the water flow path thereby to improve heat transfer and terminating in a gas outlet, the combustion chamber comprising at least one modular element, joined axially to the frustro-conical cover and coaxial therewith. The modular element comprises an inner ring and means of defining the circumferential, radial, and spiral flow paths of the hot gases.

DASS: efficient discovery and p-value calculation of substructures in unordered data.

PubMed

Hollunder, Jens; Friedel, Maik; Beyer, Andreas; Workman, Christopher T; Wilhelm, Thomas

2007-01-01

Pattern identification in biological sequence data is one of the main objectives of bioinformatics research. However, few methods are available for detecting patterns (substructures) in unordered datasets. Data mining algorithms mainly developed outside the realm of bioinformatics have been adapted for that purpose, but typically do not determine the statistical significance of the identified patterns. Moreover, these algorithms do not exploit the often modular structure of biological data. We present the algorithm DASS (Discovery of All Significant Substructures) that first identifies all substructures in unordered data (DASS(Sub)) in a manner that is especially efficient for modular data. In addition, DASS calculates the statistical significance of the identified substructures, for sets with at most one element of each type (DASS(P(set))), or for sets with multiple occurrence of elements (DASS(P(mset))). The power and versatility of DASS is demonstrated by four examples: combinations of protein domains in multi-domain proteins, combinations of proteins in protein complexes (protein subcomplexes), combinations of transcription factor target sites in promoter regions and evolutionarily conserved protein interaction subnetworks. The program code and additional data are available at http://www.fli-leibniz.de/tsb/DASS

Modular Wideband Active Vibration Absorber

NASA Technical Reports Server (NTRS)

Zewari, Wahid; Lee, Kenneth Y.; Smith, David R.

1999-01-01

A comparison of space experiments with previous missions shows a common theme. Some of the recent experiments are based on the scientific fundamentals of instruments of prior years. However, the main distinguishing characteristic is the embodiment of advances in engineering and manufacturing in order to extract clearer and sharper images and extend the limits of measurement. One area of importance to future missions is providing vibration free observation platforms at acceptable costs. It has been shown by researchers that vibration problems cannot be eliminated by passive isolation techniques alone. Therefore, various organizations have conducted research in the area of combining active and passive vibration control techniques. The essence of this paper is to present progress in what is believed to be a new concept in this arena. It is based on the notion that if one active element in a vibration transmission path can provide a reasonable vibration attenuation, two active elements in series may provide more control options and better results. The paper presents the functions of a modular split shaft linear actuator developed by NASA's Goddard Space Flight Center and University of Massachusetts Lowell. It discusses some of the control possibilities facilitated by the device. Some preliminary findings and problems are also discussed.

Smart Antenna UKM Testbed for Digital Beamforming System

NASA Astrophysics Data System (ADS)

Islam, Mohammad Tariqul; Misran, Norbahiah; Yatim, Baharudin

2009-12-01

A new design of smart antenna testbed developed at UKM for digital beamforming purpose is proposed. The smart antenna UKM testbed developed based on modular design employing two novel designs of L-probe fed inverted hybrid E-H (LIEH) array antenna and software reconfigurable digital beamforming system (DBS). The antenna is developed based on using the novel LIEH microstrip patch element design arranged into [InlineEquation not available: see fulltext.] uniform linear array antenna. An interface board is designed to interface to the ADC board with the RF front-end receiver. The modular concept of the system provides the capability to test the antenna hardware, beamforming unit, and beamforming algorithm in an independent manner, thus allowing the smart antenna system to be developed and tested in parallel, hence reduces the design time. The DBS was developed using a high-performance [InlineEquation not available: see fulltext.] floating-point DSP board and a 4-channel RF front-end receiver developed in-house. An interface board is designed to interface to the ADC board with the RF front-end receiver. A four-element receiving array testbed at 1.88-2.22 GHz frequency is constructed, and digital beamforming on this testbed is successfully demonstrated.

Atomically precise arrays of fluorescent silver clusters: a modular approach for metal cluster photonics on DNA nanostructures.

PubMed

Copp, Stacy M; Schultz, Danielle E; Swasey, Steven; Gwinn, Elisabeth G

2015-03-24

The remarkable precision that DNA scaffolds provide for arraying nanoscale optical elements enables optical phenomena that arise from interactions of metal nanoparticles, dye molecules, and quantum dots placed at nanoscale separations. However, control of ensemble optical properties has been limited by the difficulty of achieving uniform particle sizes and shapes. Ligand-stabilized metal clusters offer a route to atomically precise arrays that combine desirable attributes of both metals and molecules. Exploiting the unique advantages of the cluster regime requires techniques to realize controlled nanoscale placement of select cluster structures. Here we show that atomically monodisperse arrays of fluorescent, DNA-stabilized silver clusters can be realized on a prototypical scaffold, a DNA nanotube, with attachment sites separated by <10 nm. Cluster attachment is mediated by designed DNA linkers that enable isolation of specific clusters prior to assembly on nanotubes and preserve cluster structure and spectral purity after assembly. The modularity of this approach generalizes to silver clusters of diverse sizes and DNA scaffolds of many types. Thus, these silver cluster nano-optical elements, which themselves have colors selected by their particular DNA templating oligomer, bring unique dimensions of control and flexibility to the rapidly expanding field of nano-optics.

Mobile group II intron based gene targeting in Lactobacillus plantarum WCFS1.

PubMed

Sasikumar, Ponnusamy; Paul, Eldho; Gomathi, Sivasamy; Abhishek, Albert; Sasikumar, Sundaresan; Selvam, Govindan Sadasivam

2016-10-01

The usage of recombinant lactic acid bacteria for delivery of therapeutic proteins to the mucosa has been emerging. In the present study, an attempt was made to engineer a thyA mutant of Lactobacillus plantarum (L. plantarum) using lactococcal group II intron Ll.LtrB for the development of biologically contained recombinant L. plantarum for prevention of calcium oxalate stone disease. The 3 kb Ll.LtrB intron donor cassettes from the source vector pACD4C was PCR amplified, ligated into pSIP series of lactobacillus vector pLp_3050sAmyA, yielding a novel vector pLpACD4C (8.6 kb). The quantitative real-time PCR experiment shows 94-fold increased expression of Ll.LtrB intron and 14-fold increased expression of ltrA gene in recombinant L. plantarum containing pLpACD4C. In order to target the thyA gene, the potential intron RNA binding sites in the thyA gene of L. plantarum was predicted with help of computer algorithm. The insertion location 188|189s of thyA gene (lowest E-0.134) was chosen and the wild type intron Ll.LtrB was PCR modified, yielding a retargeted intron of pLpACDthyA. The retargeted intron was expressed by using induction peptide (sppIP), subsequently the integration of intron in thyA gene was identified by PCR screening and finally ThyA - mutant of L. plantarum (ThyA18) was detected. In vitro growth curve result showed that in the absence of thymidine, colony forming units of mutant ThyA18 was decreased, whereas high thymidine concentration (10 μM) supported the growth of the culture until saturation. In conclusion, ThyA - mutant of L. plantarum (ThyA18) constructed in this study will be used as a biologically contained recombinant probiotic to deliver oxalate decarboxylase into the lumen for treatment of hyperoxaluria and calcium oxalate stone deposition. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rigid porous filter

DOEpatents

Chiang, Ta-Kuan; Straub, Douglas L.; Dennis, Richard A.

2000-01-01

The present invention involves a porous rigid filter including a plurality of concentric filtration elements having internal flow passages and forming external flow passages there between. The present invention also involves a pressure vessel containing the filter for the removal of particulates from high pressure particulate containing gases, and further involves a method for using the filter to remove such particulates. The present filter has the advantage of requiring fewer filter elements due to the high surface area-to-volume ratio provided by the filter, requires a reduced pressure vessel size, and exhibits enhanced mechanical design properties, improved cleaning properties, configuration options, modularity and ease of fabrication.

Seakeeping with the semi-Lagrangian particle finite element method

NASA Astrophysics Data System (ADS)

Nadukandi, Prashanth; Servan-Camas, Borja; Becker, Pablo Agustín; Garcia-Espinosa, Julio

2017-07-01

The application of the semi-Lagrangian particle finite element method (SL-PFEM) for the seakeeping simulation of the wave adaptive modular vehicle under spray generating conditions is presented. The time integration of the Lagrangian advection is done using the explicit integration of the velocity and acceleration along the streamlines (X-IVAS). Despite the suitability of the SL-PFEM for the considered seakeeping application, small time steps were needed in the X-IVAS scheme to control the solution accuracy. A preliminary proposal to overcome this limitation of the X-IVAS scheme for seakeeping simulations is presented.

Optical design of MOEMS-based micro-mechatronic modules for applications in spectroscopy

NASA Astrophysics Data System (ADS)

Tortschanoff, A.; Kremer, M.; Sandner, T.; Kenda, A.

2014-05-01

One of the important challenges for widespread application of MOEMS devices is to provide a modular interface for easy handling and accurate driving of the MOEMS elements, in order to enable seamless integration in larger spectroscopic system solutions. In this contribution we present in much detail the optical design of MOEMS driver modules comprising optical position sensing together with driver electronics, which can actively control different electrostatically driven MOEMS. Furthermore we will present concepts for compact spectroscopic devices, based on different MOEMS scanner modules with lD and 2D optical elements.

Mikado: A graphic program

NASA Astrophysics Data System (ADS)

Secretan, Y.

A discussion of the modular program Mikado is presented. Mikado was developed with the goal of creating a flexible graphic tool to display and help analyze the results of finite element fluid flow computations. Mikado works on unstructured meshes, with elements of mixed geometric type, but also offers the possibility of using structured meshes. The program can be operated by both menu and mouse (interactive), or by command file (batch). Mikado is written in FORTRAN, except for a few system dependent subroutines which are in C. It runs presently on Silicon Graphics' workstations and could be easily ported to the IBM-RISC System/6000 family of workstations.

Convectively cooled electrical grid structure

DOEpatents

Paterson, James A.; Koehler, Gary W.

1982-01-01

Undesirable distortions of electrical grid conductors (12) from thermal cycling are minimized and related problems such as unwanted thermionic emission and structural failure from overheating are avoided by providing for a flow of fluid coolant within each conductor (12). The conductors (12) are secured at each end to separate flexible support elements (16) which accommodate to individual longitudinal expansion and contraction of each conductor (12) while resisting lateral displacements, the coolant flow preferably being directed into and out of each conductor through passages (48) in the flexible support elements (16). The grid (11) may have a modular or divided construction which facilitates manufacture and repairs.

Proliferation of group II introns in the chloroplast genome of the green alga Oedocladium carolinianum (Chlorophyceae).

PubMed

Brouard, Jean-Simon; Turmel, Monique; Otis, Christian; Lemieux, Claude

2016-01-01

The chloroplast genome sustained extensive changes in architecture during the evolution of the Chlorophyceae, a morphologically and ecologically diverse class of green algae belonging to the Chlorophyta; however, the forces driving these changes are poorly understood. The five orders recognized in the Chlorophyceae form two major clades: the CS clade consisting of the Chlamydomonadales and Sphaeropleales, and the OCC clade consisting of the Oedogoniales, Chaetophorales, and Chaetopeltidales. In the OCC clade, considerable variations in chloroplast DNA (cpDNA) structure, size, gene order, and intron content have been observed. The large inverted repeat (IR), an ancestral feature characteristic of most green plants, is present in Oedogonium cardiacum (Oedogoniales) but is lacking in the examined members of the Chaetophorales and Chaetopeltidales. Remarkably, the Oedogonium 35.5-kb IR houses genes that were putatively acquired through horizontal DNA transfer. To better understand the dynamics of chloroplast genome evolution in the Oedogoniales, we analyzed the cpDNA of a second representative of this order, Oedocladium carolinianum . The Oedocladium cpDNA was sequenced and annotated. The evolutionary distances separating Oedocladium and Oedogonium cpDNAs and two other pairs of chlorophycean cpDNAs were estimated using a 61-gene data set. Phylogenetic analysis of an alignment of group IIA introns from members of the OCC clade was performed. Secondary structures and insertion sites of oedogonialean group IIA introns were analyzed. The 204,438-bp Oedocladium genome is 7.9 kb larger than the Oedogonium genome, but its repertoire of conserved genes is remarkably similar and gene order differs by only one reversal. Although the 23.7-kb IR is missing the putative foreign genes found in Oedogonium , it contains sequences coding for a putative phage or bacterial DNA primase and a hypothetical protein. Intergenic sequences are 1.5-fold longer and dispersed repeats are more abundant, but a smaller fraction of the Oedocladium genome is occupied by introns. Six additional group II introns are present, five of which lack ORFs and carry highly similar sequences to that of the ORF-less IIA intron shared with Oedogonium . Secondary structure analysis of the group IIA introns disclosed marked differences in the exon-binding sites; however, each intron showed perfect or nearly perfect base pairing interactions with its target site. Our results suggest that chloroplast genes rearrange more slowly in the Oedogoniales than in the Chaetophorales and raise questions as to what was the nature of the foreign coding sequences in the IR of the common ancestor of the Oedogoniales. They provide the first evidence for intragenomic proliferation of group IIA introns in the Viridiplantae, revealing that intron spread in the Oedocladium lineage likely occurred by retrohoming after sequence divergence of the exon-binding sites.

Localized Triple Modular Redundancy vs. Distributed Triple Modular Redundancy on a ProASIC3E Reprogrammable FPGA

NASA Technical Reports Server (NTRS)

McGuffey, Alex; Berg, Melanie; Pellish, Jonathan

2010-01-01

Field programmable gate arrays (FPGA) are used in every space application. Currently, most space flight applications use radiation hardened (RH) FPGAs, which are very expensive. There is a desire to use cheaper, commercial off the shelf reprogrammable FPGAs, which are more susceptible to radiation effects known as single-event effects (SEE). The RH parts have SEE and total ionizing dose (TID) hardened elements pre-integrated into the part. This means that the designer does not need to implement any hardening techniques while configuring the device. The COTS parts on the other hand must be mitigated by design in order to insure any form of mitigation. The design techniques this project examines concern the use of localized triple modular redundancy (LTMR) and distributed triple modular redundancy (DTMR). LTMR triples every flip flop in the device architecture while DTMR triples everything except for the global routes (clocks, resets, and enables). The testing was performed on a ProASIC3E FPGA at the Texas A&M cyclotron facility. Two design architectures were used: shift registers and counters, both with LTMR and DTMR mitigation techniques. The test results prove that DTMR is more effective at reducing SEE than LTMR. We also determined that there was not a significant difference between the use of shift registers and counters for test purposes. More testing is required to obtain additional linear energy transfer values for each architecture and mitigation technique in order to determine the most cost-effective method of SEE mitigation.

Using modular psychotherapy in school mental health: Provider perspectives on intervention-setting fit

PubMed Central

Lyon, Aaron R.; Ludwig, Kristy; Romano, Evalynn; Koltracht, Jane; Stoep, Ann Vander; McCauley, Elizabeth

2013-01-01

Objective The “fit” or appropriateness of well-researched interventions within usual care contexts is among the most commonly-cited, but infrequently researched, factors in the successful implementation of new practices. The current study was initiated to address two exploratory research questions: (1) How do clinicians describe their current school mental health service delivery context? and (2) How do clinicians describe the fit between modular psychotherapy and multiple levels of the school mental health service delivery context? Method Following a year-long training and consultation program in an evidence-based, modular approach to psychotherapy, semi-structured qualitative interviews were conducted with seventeen school-based mental health providers to evaluate their perspectives on the appropriateness of implementing the approach within a system of school-based health centers. Interviews were transcribed and coded for themes using conventional and directed content analysis. Results Findings identified key elements of the school mental health context including characteristics of the clinicians, their practices, the school context, and the service recipients. Specific evaluation of intervention-setting appropriateness elicited many comments about both practical and value-based (e.g., cultural considerations) aspects at the clinician and client levels, but fewer comments at the school or organizational levels. Conclusions Results suggest that a modular approach may fit well with the school mental health service context, especially along practical aspects of appropriateness. Future research focused on the development of methods for routinely assessing appropriateness at different stages of the implementation process is recommended. PMID:24134063