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.

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.

A deep intronic mutation in the SLC12A3 gene leads to Gitelman syndrome.

PubMed

Nozu, Kandai; Iijima, Kazumoto; Nozu, Yoshimi; Ikegami, Ei; Imai, Takehide; Fu, Xue Jun; Kaito, Hiroshi; Nakanishi, Koichi; Yoshikawa, Norishige; Matsuo, Masafumi

2009-11-01

Many mutations have been detected in the SLC12A3 gene of Gitelman syndrome (GS, OMIM 263800) patients. In previous studies, only one mutant allele was detected in approximately 20 to 41% of patients with GS; however, the exact reason for the nonidentification has not been established. In this study, we used RT-PCR using mRNA to investigate for the first time transcript abnormalities caused by deep intronic mutation. Direct sequencing analysis of leukocyte DNA identified one base insertion in exon 6 (c.818_819insG), but no mutation was detected in another allele. We analyzed RNA extracted from leukocytes and urine sediments and detected unknown sequence containing 238bp between exons 13 and 14. The genomic DNA analysis of intron 13 revealed a single-base substitution (c.1670-191C>T) that creates a new donor splice site within the intron resulting in the inclusion of a novel cryptic exon in mRNA. This is the first report of creation of a splice site by a deep intronic single-nucleotide change in GS and the first report to detect the onset mechanism in a patient with GS and missing mutation in one allele. This molecular onset mechanism may partly explain the poor success rate of mutation detection in both alleles of patients with GS.

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

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.

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

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

A SMN-Dependent U12 Splicing Event Essential for Motor Circuit Function

PubMed Central

Lotti, Francesco; Imlach, Wendy L.; Saieva, Luciano; Beck, Erin S.; Hao, Le T.; Li, Darrick K.; Jiao, Wei; Mentis, George Z.; Beattie, Christine E.; McCabe, Brian D.; Pellizzoni, Livio

2012-01-01

SUMMARY Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a novel protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA. PMID:23063131

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

PubMed Central

Jansen, Robert K.; Wojciechowski, Martin F.; Sanniyasi, Elumalai; Lee, Seung-Bum; Daniell, Henry

2008-01-01

Chickpea (Cicer arietinum, Leguminosae), an important grain legume, is widely used for food and fodder throughout the world. We sequenced the complete plastid genome of chickpea, which is 125,319 bp in size, and contains only one copy of the inverted repeat (IR). The genome encodes 108 genes, including 4 rRNAs, 29 tRNAs, and 75 proteins. The genes rps16, infA, and ycf4 are absent in the chickpea plastid genome, and ndhB has an internal stop codon in the 5′exon, similar to other legumes. Two genes have lost their introns, one in the 3′exon of the transpliced gene rps12, and the one between exons 1 and 2 of clpP; this represents the first documented case of the loss of introns from both of these genes in the same plastid genome. An extensive phylogenetic survey of these intron losses was performed on 302 taxa across legumes and the related family Polygalaceae. The clpP intron has been lost exclusively in taxa from the temperate “IR-lacking clade” (IRLC), whereas the rps12 intron has been lost in most members of the IRLC (with the exception of Wisteria, Callerya, Afgekia, and certain species of Millettia, which represent the earliest diverging lineages of this clade), and in the tribe Desmodieae, which is closely related to the tribes Phaseoleae and Psoraleeae. Data provided here suggest that the loss of the rps12 intron occurred after the loss of the IR. The two new genomic changes identified in the present study provide additional support of the monophyly of the IR-loss clade, and resolution of the pattern of the earliest-branching lineages in this clade. The availability of the complete chickpea plastid genome sequence also provides valuable information on intergenic spacer regions among legumes and endogenous regulatory sequences for plastid genetic engineering. PMID:18638561

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

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

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.

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

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.

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.

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

Typing of artiodactyl MHC-DRB genes with the help of intronic simple repeated DNA sequences.

PubMed

Schwaiger, F W; Buitkamp, J; Weyers, E; Epplen, J T

1993-02-01

An efficient oligonucleotide typing method for the highly polymorphic MHC-DRB genes is described for artiodactyls like cattle, sheep and goat. By means of the polymerase chain reaction, the second exon of MHC-DRB is amplified as well as part of the adjacent intron containing a mixed simple repeat sequence. Using this primer combination we were able to amplify the MHC-DRB exons 2 and adjacent introns from all of the investigated 10 species of the family of Bovidae and giraffes. Therefore, the DRB genes of novel artiodactyl species can also be readily studied. Oligonucleotide probes specific for the polymorphisms of ungulate DRB genes are used with which sequences differing in at least one single base can be distinguished. Exonic polymorphism was found to be correlated with the allele lengths and the patterns of the repeat structures. Hence oligonucleotide probes specific for different simple repeats and polymorphic positions serve also for typing across species barriers. The strict correlation of sequence length and exonic polymorphism permits a preselection of specific oligonucleotides for hybridization. Thus more than 20 alleles can already be differentiated from each of the three species.

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.

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

PubMed Central

Jangi, Mohini; Fleet, Christina; Cullen, Patrick; Gupta, Shipra V.; Mekhoubad, Shila; Chiao, Eric; Allaire, Norm; Bennett, C. Frank; Rigo, Frank; Krainer, Adrian R.; Hurt, Jessica A.; Carulli, John P.; Staropoli, John F.

2017-01-01

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death. PMID:28270613

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

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

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.

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

PubMed

Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

2015-08-01

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

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.

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

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.

Intronic splicing mutations in PTCH1 cause Gorlin syndrome.

PubMed

Bholah, Zaynab; Smith, Miriam J; Byers, Helen J; Miles, Emma K; Evans, D Gareth; Newman, William G

2014-09-01

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

PubMed

Pendleton, Kathryn E; Chen, Beibei; Liu, Kuanqing; Hunter, Olga V; Xie, Yang; Tu, Benjamin P; Conrad, Nicholas K

2017-05-18

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N 6 -adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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.

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.

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

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.

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

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

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.

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.

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

A base substitution in the donor site of intron 12 of KIT gene is responsible for the dominant white coat colour of blue fox (Alopex lagopus).

PubMed

Yan, S Q; Hou, J N; Bai, C Y; Jiang, Y; Zhang, X J; Ren, H L; Sun, B X; Zhao, Z H; Sun, J H

2014-04-01

The dominant white coat colour of farmed blue fox is inherited as a monogenic autosomal dominant trait and is suggested to be embryonic lethal in the homozygous state. In this study, the transcripts of KIT were identified by RT-PCR for a dominant white fox and a normal blue fox. Sequence analysis showed that the KIT transcript in normal blue fox contained the full-length coding sequence of 2919 bp (GenBank Acc. No KF530833), but in the dominant white individual, a truncated isoform lacking the entire exon 12 specifically co-expressed with the normal transcript. Genomic DNA sequencing revealed that a single nucleotide polymorphism (c.1867+1G>T) in intron 12 appeared only in the dominant white individuals and a 1-bp ins/del polymorphism in the same intron showed in individuals representing two different coat colours. Genotyping results of the SNP with PCR-RFLP in 185 individuals showed all 90 normal blue foxes were homozygous for the G allele, and all dominant white individuals were heterozygous. Due to the truncated protein with a deletion of 35 amino acids and an amino acid replacement (p.Pro623Ala) located in the conserved ATP binding domain, we propose that the mutant receptor had absent tyrosine kinase activity. These findings reveal that the base substitution at the first nucleotide of intron 12 of KIT gene, resulting in skipping of exon 12, is a causative mutation responsible for the dominant white phenotype of blue fox. © 2013 Stichting International Foundation for Animal Genetics.

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.

G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

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

Willing, M.; Deschenes, S.

We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exonmore » 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.« less

A novel type of EWS-CHOP fusion gene in myxoid liposarcoma

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

Matsui, Yoshito; Ueda, Takafumi; Kubo, Takahiro

2006-09-22

The cytogenetic hallmark of myxoid type and round cell type liposarcoma consists of reciprocal translocation of t(12;16)(q13;p11) and t(12;22)(q13;q12), which results in fusion of TLS/FUS and CHOP, and EWS and CHOP, respectively. Nine structural variations of the TLS/FUS-CHOP chimeric transcript have been reported, however, only two types of EWS-CHOP have been described. We describe here a case of myxoid liposarcoma containing a novel EWS-CHOP chimeric transcript and identified the breakpoint occurring in intron 13 of EWS. Reverse transcription-polymerase chain reaction and direct sequence showed that exon 13 of EWS was in-frame fused to exon 2 of CHOP. Genomic analysis revealedmore » that the breaks were located in intron 13 of EWS and intron 1 of CHOP.« less

Another heritage from the RNA world: self-excision of intron sequence from nuclear pre-tRNAs.

PubMed

Weber, U; Beier, H; Gross, H J

1996-06-15

The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7 transcript corresponding to a pre-tRNA(Tyr) from Arabidopsis thaliana has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3'-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5'-splice site. The reaction results in fragments with 2',3'-cyclic phosphate and 5'-OH termini. It is resistant to proteinase K and/or SDS treatment and is not inhibited by added tRNA. The self-cleavage depends on Mg2+ and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed for autolytic intron excision and, in parallel, for enzymatic in vitro splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than the enzymatic intron excision in a wheat germ S23 extract, we propose that the splicing endonuclease may function by improving the preciseness and efficiency of an inherent pre-tRNA self-cleavage activity.

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

PubMed

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

2018-06-15

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

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

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

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

Protein arginine methylation of Npl3 promotes splicing of the SUS1 intron harboring non-consensus 5' splice site and branch site.

PubMed

Muddukrishna, Bhavana; Jackson, Christopher A; Yu, Michael C

2017-06-01

Protein arginine methylation occurs on spliceosomal components and spliceosome-associated proteins, but how this modification contributes to their function in pre-mRNA splicing remains sparse. Here we provide evidence that protein arginine methylation of the yeast SR-/hnRNP-like protein Npl3 plays a role in facilitating efficient splicing of the SUS1 intron that harbors a non-consensus 5' splice site and branch site. In yeast cells lacking the major protein arginine methyltransferase HMT1, we observed a change in the co-transcriptional recruitment of the U1 snRNP subunit Snp1 and Npl3 to pre-mRNAs harboring both consensus (ECM33 and ASC1) and non-consensus (SUS1) 5' splice site and branch site. Using an Npl3 mutant that phenocopies wild-type Npl3 when expressed in Δhmt1 cells, we showed that the arginine methylation of Npl3 is responsible for this. Examination of pre-mRNA splicing efficiency in these mutants reveals the requirement of Npl3 methylation for the efficient splicing of SUS1 intron 1, but not of ECM33 or ASC1. Changing the 5' splice site and branch site in SUS1 intron 1 to the consensus form restored splicing efficiency in an Hmt1-independent manner. Results from biochemical studies show that methylation of Npl3 promotes its optimal association with the U1 snRNP through its association with the U1 snRNP subunit Mud1. Based on these data, we propose a model in which Hmt1, via arginine methylation of Npl3, facilitates U1 snRNP engagement with the pre-mRNA to promote usage of non-consensus splice sites by the splicing machinery. Published by Elsevier B.V.

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16.

PubMed

Martínez-Salazar, Martha; López-Urrutia, Eduardo; Arechaga-Ocampo, Elena; Bonilla-Moreno, Raul; Martínez-Castillo, Macario; Díaz-Hernández, Job; Del Moral-Hernández, Oscar; Cedillo-Barrón, Leticia; Martines-Juarez, Víctor; De Nova-Ocampo, Monica; Valdes, Jesús; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2014-12-05

The human papillomavirus type 16 (HPV-16) E6/E7 spliced transcripts are heterogeneously expressed in cervical carcinoma. The heterogeneity of the E6/E7 splicing profile might be in part due to the intrinsic variation of splicing factors in tumor cells. However, the splicing factors that bind the E6/E7 intron 1 (In-1) have not been defined. Therefore, we aimed to identify these factors; we used HeLa nuclear extracts (NE) for in vitro spliceosome assembly. The proteins were allowed to bind to an RNA/DNA hybrid formed by the In-1 transcript and a 5'-biotinylated DNA oligonucleotide complementary to the upstream exon sequence, which prevented interference in protein binding to the intron. The hybrid probes bound with the nuclear proteins were coupled to streptavidin magnetic beads for chromatography affinity purification. Proteins were eluted and identified by mass spectrometry (MS). Approximately 170 proteins were identified by MS, 80% of which were RNA binding proteins, including canonical spliceosome core components, helicases and regulatory splicing factors. The canonical factors were identified as components of the spliceosomal B-complex. Although 35-40 of the identified factors were cognate splicing factors or helicases, they have not been previously detected in spliceosome complexes that were assembled using in vivo or in vitro models. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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

Defective minor spliceosome mRNA processing results in isolated familial growth hormone deficiency

PubMed Central

Argente, Jesús; Flores, Raquel; Gutiérrez-Arumí, Armand; Verma, Bhupendra; Martos-Moreno, Gabriel Á; Cuscó, Ivon; Oghabian, Ali; Chowen, Julie A; Frilander, Mikko J; Pérez-Jurado, Luis A

2014-01-01

The molecular basis of a significant number of cases of isolated growth hormone deficiency remains unknown. We describe three sisters affected with severe isolated growth hormone deficiency and pituitary hypoplasia caused by biallelic mutations in the RNPC3 gene, which codes for a minor spliceosome protein required for U11/U12 small nuclear ribonucleoprotein (snRNP) formation and splicing of U12-type introns. We found anomalies in U11/U12 di-snRNP formation and in splicing of multiple U12-type introns in patient cells. Defective transcripts include preprohormone convertases SPCS2 and SPCS3 and actin-related ARPC5L genes, which are candidates for the somatotroph-restricted dysfunction. The reported novel mechanism for familial growth hormone deficiency demonstrates that general mRNA processing defects of the minor spliceosome can lead to very narrow tissue-specific consequences. Subject Categories Genetics, Gene Therapy ' Genetic Disease; Metabolism PMID:24480542

Association analysis of the functional MAOA gene promoter and MAOB gene intron 13 polymorphisms in tension type headache patients.

PubMed

Edgnülü, Tuba G; Özge, Aynur; Erdal, Nurten; Kuru, Oktay; Erdal, Mehmet E

2014-01-01

Monoamine oxidase (MAO) enzymes play an important role in the etiology of many neurological diseases. Tension type headache (TTH) treatments contain inhibitors for selective re-uptake of serotonin and monoamine oxidase inhibitors. MAO (EC 1.4.3.4) has two isoenzymes known as MAOA and MAOB. A promoter polymorphism of a variable number of tandem repeats (VNTR) in the MAOA gene seems to affect MAOA transcriptional activity in vitro. Also, G/A polymorphism in intron 13 (rs1799836) of the MAOB gene have been previously found to be associated with the variability of MAOB enzyme activity. The aim of our study was to investigate a possible association of monoamine oxidase (MAOA and MAOB) gene polymorphisms in tension type headache. MAO gene polymorphisms were examined in a group of 120 TTH patients and in another 168 unrelated healthy volunteers (control group). MAOA promoter and MAOB intron 13 polymorphisms were genotyped using PCR-based methods. An overall comparison between the genotype of MAOA and MAOB genes and allele frequencies of the patients and the control group did not reveal any statistically significant difference between the patients and the control group (p=0.162). Factors like estrogen dosage, the limited number of male patients and other genes' neurotransmitters involved in the etiology of TTH could be responsible for our non-significant results.

Intronic deletions in the SLC34A3 gene: A cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria

PubMed Central

Ichikawa, Shoji; Tuchman, Shamir; Padgett, Leah R.; Gray, Amie K.; Baluarte, H. Jorge; Econs, Michael J.

2013-01-01

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6 ½-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24- hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH) levels, and elevated 1,25(OH)2D level. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440–1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63 bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH. PMID:24176905

Intronic deletions in the SLC34A3 gene: a cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria.

PubMed

Ichikawa, Shoji; Tuchman, Shamir; Padgett, Leah R; Gray, Amie K; Baluarte, H Jorge; Econs, Michael J

2014-02-01

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6-1/2-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24-hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH), and elevated 1,25(OH)2D. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440-1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH. © 2013.

Geological investigations in the U12b.03 and U12b.04 tunnels, Nevada Test Site

USGS Publications Warehouse

Diment, W.H.; Wilmarth, V. R.; McKeown, F.A.; Dickey, D.D.; Hinrichs, E.N.; Botinelly, T.; Roach, C.H.; Byers, F.M.; Hawley, C.C.; Izett, G.A.; Clebsch, Alfred

1959-01-01

The U12b.03 and .04 tunnels are part of the U12b (Rainier) tunnel complex that was driven northwestward from the steep east slope of Rainier Mesa (a prominent topographic feature in the northwest part of the Test Site (fig . 2)). The U12b.03 tunnel trends north from a point about 980 feet from the portal of the U12b tunnel (fig. 3). The U12b.03 tunnel consists of 620 feet of tunnel, two alcoves, and a shot chamber. The tunnel is irregular, ranging from 6 to 10 feet in width, and 6 to 9 feet in height. The shot chamber at the north end of the tunnel is 22 feet on each sidee The vertical and minimum cover over the shot chamber are 610 and 510 feet, respectively.

Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors.

PubMed

Wickramasinghe, Susiji; Yatawara, Lalani; Nagataki, Mitsuru; Agatsuma, Takeshi

2016-10-01

To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis). Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K m value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K m value of the mutant (Serine to Glycine) increased to 0.19 mM. The K m value (0.19 mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A. indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Arginine kinase in T. canis has a seven-exon/six-intron gene

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.

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.

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.

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

Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution

PubMed Central

Lambowitz, Alan M.; Belfort, Marlene

2015-01-01

SUMMARY This review focuses on recent developments in our understanding of group II intron function, the relationships of these introns to retrotransposons and spliceosomes, and how their common features have informed thinking about bacterial group II introns as key elements in eukaryotic evolution. Reverse transcriptase-mediated and host factor-aided intron retrohoming pathways are considered along with retrotransposition mechanisms to novel sites in bacteria, where group II introns are thought to have originated. DNA target recognition and movement by target-primed reverse transcription infer an evolutionary relationship among group II introns, non-LTR retrotransposons, such as LINE elements, and telomerase. Additionally, group II introns are almost certainly the progenitors of spliceosomal introns. Their profound similarities include splicing chemistry extending to RNA catalysis, reaction stereochemistry, and the position of two divalent metals that perform catalysis at the RNA active site. There are also sequence and structural similarities between group II introns and the spliceosome’s small nuclear RNAs (snRNAs) and between a highly conserved core spliceosomal protein Prp8 and a group II intron-like reverse transcriptase. It has been proposed that group II introns entered eukaryotes during bacterial endosymbiosis or bacterial-archaeal fusion, proliferated within the nuclear genome, necessitating evolution of the nuclear envelope, and fragmented giving rise to spliceosomal introns. Thus, these bacterial self-splicing mobile elements have fundamentally impacted the composition of extant eukaryotic genomes, including the human genome, most of which is derived from close relatives of mobile group II introns. PMID:25878921

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.

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.

Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution.

PubMed

Zhao, Chen; Pyle, Anna Marie

2016-06-01

Group II introns are self-splicing ribozymes that are essential in many organisms, and they have been hypothesized to share a common evolutionary ancestor with the spliceosome. Although structural similarity of RNA components supports this connection, it is of interest to determine whether associated protein factors also share an evolutionary heritage. Here we present the crystal structures of reverse transcriptase (RT) domains from two group II intron-encoded proteins (maturases) from Roseburia intestinalis and Eubacterium rectale, obtained at 1.2-Å and 2.1-Å resolution, respectively. These domains are more similar in architecture to the spliceosomal Prp8 RT-like domain than to any other RTs, and they share substantial similarity with flaviviral RNA polymerases. The RT domain itself is sufficient for binding intron RNA with high affinity and specificity, and it is contained within an active RT enzyme. These studies provide a foundation for understanding structure-function relationships within group II intron-maturase complexes.

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.

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome

PubMed Central

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes. PMID:25482895

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome.

PubMed

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes.

Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA

PubMed Central

Howe, Kenneth James; Ares, Manuel

1997-01-01

Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA–RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5′ splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5′ splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing. PMID:9356473

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

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.

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.

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.

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

Structural and Functional Characterization of Ribosomal Protein Gene Introns in Sponges

PubMed Central

Perina, Drago; Korolija, Marina; Mikoč, Andreja; Roller, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

2012-01-01

Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with “higher” metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales. PMID:22880015

Structural and functional characterization of ribosomal protein gene introns in sponges.

PubMed

Perina, Drago; Korolija, Marina; Mikoč, Andreja; Roller, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

2012-01-01

Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales.

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.

Effective suppression of dengue virus using a novel group-I intron that induces apoptotic cell death upon infection through conditional expression of the Bax C-terminal domain.

PubMed

Carter, James R; Keith, James H; Fraser, Tresa S; Dawson, James L; Kucharski, Cheryl A; Horne, Kate M; Higgs, Stephen; Fraser, Malcolm J

2014-06-13

Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3' exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3' exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death. This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3' exon that trans-splices conserved sequences of the 5' CS

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

[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.

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.

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

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.

Mitochondrial intronic open reading frames in Podospora: mobility and consecutive exonic sequence variations.

PubMed

Sellem, C H; d'Aubenton-Carafa, Y; Rossignol, M; Belcour, L

1996-06-01

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group 1 intronic ORFs are mobile elements and that their transfer, and concomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.

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

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

PubMed

Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

2015-07-15

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

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

Characterization of the intronic portion of cadherin superfamily members, common cancer orchestrators

PubMed Central

Oliveira, Patrícia; Sanges, Remo; Huntsman, David; Stupka, Elia; Oliveira, Carla

2012-01-01

Cadherins are cell–cell adhesion proteins essential for the maintenance of tissue architecture and integrity, and their impairment is often associated with human cancer. Knowledge regarding regulatory mechanisms associated with cadherin misexpression in cancer is scarce. Specific features of the intronic-structure and intronic-based regulatory mechanisms in the cadherin superfamily are unidentified. This study aims at systematically characterizing the intronic portion of cadherin superfamily members and the identification of intronic regions constituting putative targets/triggers of regulation, using a bioinformatic approach and biological data mining. Our study demonstrates that the cadherin superfamily genes harbour specific characteristics in comparison to all non-cadherin genes, both from the genomic and transcriptional standpoints. Cadherin superfamily genes display higher average total intron number and significantly longer introns than other genes and across the entire vertebrate lineage. Moreover, in the human genome, we observed an uncommon high frequency of MIR (mammalian-wide interspersed repeats) and MaLR (mammalian-wide interspersed repeats, a subtype of LTR) regulatory-associated repetitive elements at 5′-located introns, concomitantly with increased de novo intronic transcription. Using this approach, we identified cadherin intronic-specific sites that may constitute novel targets/triggers of cadherin superfamily expression regulation. These findings pinpoint the need to identify mechanisms affecting particularly MIR and MaLR elements located in introns 2 and 3 of human cadherin genes, possibly important in the expression modulation of this superfamily in homeostasis and cancer. PMID:22317972

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

Mitochondrial intronic open reading frames in Podospora: Mobility and consecutive exonic sequence variations

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

Sellem, C.H.; Rossignol, M.; Belcour, L.

1996-06-01

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optical sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences.more » In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes. 46 refs., 5 figs., 2 tabs.« less

Mitochondrial Intronic Open Reading Frames in Podospora: Mobility and Consecutive Exonic Sequence Variations

PubMed Central

Sellem, C. H.; d'Aubenton-Carafa, Y.; Rossignol, M.; Belcour, L.

1996-01-01

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes. PMID:8725226

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.

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.

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

PubMed Central

Piazza, Carol Lyn; Smith, Dorie

2018-01-01

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting.

PubMed

Qu, Guosheng; Piazza, Carol Lyn; Smith, Dorie; Belfort, Marlene

2018-06-15

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis , inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. © 2018, Qu et al.

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

Crystal structure and physical properties of a novel Kondo antiferromagnet: U3Ru4Al12

NASA Astrophysics Data System (ADS)

Pasturel, M; Tougait, O; Potel, M; Roisnel, T; Wochowski, K; Noël, H; Troć, R

2009-03-01

A novel ternary compound U3Ru4Al12 has been identified in the U-Ru-Al ternary diagram. Single-crystal x-ray diffraction indicates a hexagonal Gd3Ru4Al12-type structure for this uranium-based intermetallic. While this structure type usually induces geometrically a spin-glass behaviour, an antiferromagnetic ordering is observed at TN = 8.4 K in the present case. The reduced effective magnetic moment of U atoms (μeff = 2.6 µB) can be explained by Kondo-like interactions and crystal field effects that have been identified by a logarithmic temperature dependence of the electrical resistivity, negative values of the magnetoresistivity and particular shape of the Seebeck coefficient.

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.

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

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.

Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

PubMed Central

Qian, Xiaoxiao; Matthews, Laura; Lightman, Stafford; Ray, David; Norman, Michael

2015-01-01

Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression. PMID:19819975

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.

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

Novel BRCA1 mutations and more frequent intron-20 alteration found among 236 women from Western Poland.

PubMed

Sobczak, K; Kozłowski, P; Napierała, M; Czarny, J; Woźniak, M; Kapuścińska, M; Lośko, M; Koziczak, M; Jasińska, A; Powierska, J; Braczkowski, R; Breborowicz, J; Godlewski, D; Mackiewicz, A; Krzyzosiak, W

1997-10-09

Three different novel BRCA1 mutations, five independent cases of the same 12 bp insertion-duplication in intron-20 and two novel rare BRCA1 sequence variants were identified among 122 Polish women with positive, in most cases moderate family history of breast and/or ovarian cancer, 80 controls and 34 unselected breast cancer tissue specimens. All mutations and variants were germline. The 4153 delA frameshift mutation, the Tyr105Cys missense mutation and two cases of the alteration in intron-20 were found in the group of healthy women with positive family history. Two other cases of the intronic insertion were found in unselected controls. Their carriers had no family history of breast or ovarian cancer but other cancers occurred in their families. The 1782 Trp/STOP nonsense mutation and one case of the insertion in intron-20 were first found in tissue specimens of breast cancer patient and breast/ovarian cancer patient, respectively. Their carriers also had no family history of breast or ovarian cancer. The distribution of the insertion in intron-20 in analysed groups and results of RT-PCR experiments suggest a less prominent role for this variant considered earlier a splicing mutation. This study shows also, that more population-oriented research is needed, involving women with less profound or even no family history of breast and ovarian cancer, to better understand the role and significance of different BRCA1 variants and mutations.

Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network

PubMed Central

Preußer, Christian; Rossbach, Oliver; Hung, Lee-Hsueh; Li, Dan; Bindereif, Albrecht

2014-01-01

Trans-splicing in trypanosomes adds a 39-nucleotide mini-exon from the spliced leader (SL) RNA to the 5′ end of each protein-coding sequence. On the other hand, cis-splicing of the few intron-containing genes requires the U1 small nuclear ribonucleoprotein (snRNP) particle. To search for potential new functions of the U1 snRNP in Trypanosoma brucei, we applied genome-wide individual-nucleotide resolution crosslinking-immunoprecipitation (iCLIP), focusing on the U1 snRNP-specific proteins U1C and U1-70K. Surprisingly, U1C and U1-70K interact not only with the U1, but also with U6 and SL RNAs. In addition, mapping of crosslinks to the cis-spliced PAP [poly(A) polymerase] pre-mRNA indicate an active role of these proteins in 5′ splice site recognition. In sum, our results demonstrate that the iCLIP approach provides insight into stable and transient RNA–protein contacts within the spliceosomal network. We propose that the U1 snRNP may represent an evolutionary link between the cis- and trans-splicing machineries, playing a dual role in 5′ splice site recognition on the trans-spliceosomal SL RNP as well as on pre-mRNA cis-introns. PMID:24748659

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

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.

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

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

Abiotic Stresses Modulate Landscape of Poplar Transcriptome via Alternative Splicing, Differential Intron Retention, and Isoform Ratio Switching

PubMed Central

Filichkin, Sergei A.; Hamilton, Michael; Dharmawardhana, Palitha D.; Singh, Sunil K.; Sullivan, Christopher; Ben-Hur, Asa; Reddy, Anireddy S. N.; Jaiswal, Pankaj

2018-01-01

Abiotic stresses affect plant physiology, development, growth, and alter pre-mRNA splicing. Western poplar is a model woody tree and a potential bioenergy feedstock. To investigate the extent of stress-regulated alternative splicing (AS), we conducted an in-depth survey of leaf, root, and stem xylem transcriptomes under drought, salt, or temperature stress. Analysis of approximately one billion of genome-aligned RNA-Seq reads from tissue- or stress-specific libraries revealed over fifteen millions of novel splice junctions. Transcript models supported by both RNA-Seq and single molecule isoform sequencing (Iso-Seq) data revealed a broad array of novel stress- and/or tissue-specific isoforms. Analysis of Iso-Seq data also resulted in the discovery of 15,087 novel transcribed regions of which 164 show AS. Our findings demonstrate that abiotic stresses profoundly perturb transcript isoform profiles and trigger widespread intron retention (IR) events. Stress treatments often increased or decreased retention of specific introns – a phenomenon described here as differential intron retention (DIR). Many differentially retained introns were regulated in a stress- and/or tissue-specific manner. A subset of transcripts harboring super stress-responsive DIR events showed persisting fluctuations in the degree of IR across all treatments and tissue types. To investigate coordinated dynamics of intron-containing transcripts in the study we quantified absolute copy number of isoforms of two conserved transcription factors (TFs) using Droplet Digital PCR. This case study suggests that stress treatments can be associated with coordinated switches in relative ratios between fully spliced and intron-retaining isoforms and may play a role in adjusting transcriptome to abiotic stresses. PMID:29483921

Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA.

PubMed

Ortiz, Raúl; Georgieva, Maya V; Gutiérrez, Sara; Pedraza, Neus; Fernández-Moya, Sandra M; Gallego, Carme

2017-07-05

Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Haplotype diversity in the equine myostatin gene with focus on variants associated with race distance propensity and muscle fiber type proportions

PubMed Central

Petersen, Jessica L; Valberg, Stephanie J; Mickelson, James R; McCue, Molly E

2014-01-01

Summary Two variants in the equine myostatin gene (MSTN), including a T/C SNP substitution in the first intron and a 227-bp SINE insertion in the promoter, are associated with muscle fiber type proportions in the Quarter Horse (QH) and with the prediction of race distance propensity in the Thoroughbred (TB). Genotypes from these loci, along with 18 additional variants surrounding MSTN, were examined in 301 horses of 14 breeds to evaluate haplotype relationships and diversity. The C allele of intron 1 was found in 12 of 14 breeds at a frequency of 0.27; the SINE was observed in five breeds, but common in only the TB and QH (0.73 and 0.48 respectively). Haplotype data suggest the SINE insertion is contemporary to and arose upon a haplotype containing the intron 1 C allele. Gluteal muscle biopsies of TBs showed a significant association of the intron 1 C allele and SINE with a higher proportion of Type 2B and lower proportion of Type 1 fibers. However, in the Belgian horse, in which the SINE is not present, the intron 1 SNP was not associated with fiber type proportions, and evaluation of fiber type proportions across the Belgian, TB and QH breeds shows the significant effect of breed on fiber type proportions is negated when evaluating horses without the SINE variant. These data suggest the SINE, rather than the intron 1 SNP, is driving the observed muscle fiber type characteristics and is the variant targeted by selection for short-distance racing. PMID:25160752

Prevalence of the intron 22 inversion of the factor VIII gene and inhibitor development in Polish patients with severe hemophilia A.

PubMed

Sawecka, Jadwiga; Skulimowska, Joanna; Windyga, Jerzy; Lopaciuk, Stanisław; Kościelak, Jerzy

2005-01-01

Patients with severe hemophilia A often develop inhibitors (antibodies) against transfused factor VIII. One hundred thirteen Polish patients with severe hemophilia A, who had been treated on demand with cryoprecipitate until 1992 and exclusively with factor VIII concentrates after 1995, were examined for intron 22 inversion by Southern blotting and the presence and magnitude of inhibitor activity in blood as determined by the Bethesda assay. The patients' ages ranged 4--67 years (mean: 33.7+/-12.4 years, median: 32 years). The number of patients with the inversion amounted to 57, while in 56 patients the mutation types were unknown; 47 patients had a distal and 10 patients a proximal type of inversion. Thirteen patients with inversions (22.8%) were found to have inhibitor in their blood. Most patients (14 out of 15) who developed inhibitors in the course of cryoprecipitate therapy were high responders. Conversely, 4 of 5 patients treated between 1992 and 1995 with both cryoprecipitate and intermediate-purity factor VIII concentrates were low responders. One multitransfused patient who had remained inhibitor-free on cryoprecipitate therapy developed inhibitor after receiving a large dose of factor VIII concentrate during surgery. None of these 5 patients developed inhibitors during their 12--40 years of treatment with cryoprecipitate, suggesting that it was less immunogenic than factor VIII concentrates. The prevalence of the intron 22 inversion mutation of the factor VIII gene in Polish hemophiliacs is similar to that in other European countries. Treatment regimens with either cryoprecipitate or virus-inactivated plasma-derived factor VIII concentrates may affect inhibitor formation in hemophilia A patients.

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.

Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis.

PubMed

Pan, Tzu-Yu; Chiou, Shyh-Shin; Wang, Chun-Chi; Wu, Shou-Mei

2014-12-01

An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 μM of YO-PRO(®)-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR. Copyright © 2014 Elsevier B.V. All rights reserved.

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

[Analysis of chloroplast rpS16 intron sequences in Lemnaceae].

PubMed

Martirosian, E V; Ryzhova, N N; Kochieva, E Z; Skriabin, K G

2009-01-01

Chloroplast rpS16 gene intron sequences were determined and characterized for twenty-five Lemnaceae accessions representing nine duckweed species. For each Lemnaceae species nucleotide substitutions and for Lemna minor, Lemna aequinoctialis, Wolffia arrhiza different indels were detected. Most of indels were found for Wolffia arrhiza and Lemna aequinoctialis. The analyses of intraspecific polymorphism resulted in identification of several gaplotypes in L. gibba and L. trisulca. Lemnaceae phylogenetic relationship based on rpS16 intron variability data has revealed significant differences between L. aequinoctialis and other Lemna species. Genetic distance values corroborated competence of Landoltia punctata separations from Spirodela into an independent generic taxon. The acceptability of rpS16 intron sequences for phylogenetic studies in Lemnaceae was shown.

Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

PubMed Central

Laurenzana, Anna; Biagioni, Alessio; D'Alessio, Silvia; Bianchini, Francesca; Chillà, Anastasia; Margheri, Francesca; Luciani, Cristina; Mazzanti, Benedetta; Pimpinelli, Nicola; Torre, Eugenio; Danese, Silvio; Calorini, Lido; Rosso, Mario Del; Fibbi, Gabriella

2014-01-01

The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of “targeted therapies” as a biological drug to be delivered directly in patient's tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis. The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a “personalized therapy”, without rejection risk. The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis. PMID:25003596

Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.

PubMed

Buzina, Alla; Lo, Mandy Y M; Moffett, Angela; Hotta, Akitsu; Fussner, Eden; Bharadwaj, Rikki R; Pasceri, Peter; Garcia-Martinez, J Victor; Bazett-Jones, David P; Ellis, James

2008-04-11

The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of

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

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.

Variants in intron 13 of the ELMO1 gene are associated with diabetic nephropathy in African Americans

PubMed Central

Leak, T. S.; Perlegas, P.S.; Smith, S.G.; Keene, K.L.; Hicks, P.J.; Langefeld, C.D.; Mychaleckyj, J.C.; Rich, S.S.; Kirk, J.K.; Freedman, B.I.; Bowden, D.W.; Sale, M.M.

2009-01-01

Variants in the engulfment and cell motility 1 (ELMO1) gene are associated with nephropathy due to type 2 diabetes mellitus (T2DM) in a Japanese cohort. We comprehensively evaluated this gene in African American (AA) T2DM patients with end-stage renal disease (ESRD). Three hundred nine HapMap tagging SNPs and 9 reportedly associated SNPs were genotyped in 577 AA T2DM-ESRD patients and 596 AA non-diabetic controls, plus 43 non-diabetic European American controls and 45 Yoruba Nigerian samples for admixture adjustment. Replication analyses were conducted in 558 AAs with T2DM-ESRD and 564 controls without diabetes. Extension analyses included 328 AA with T2DM lacking nephropathy and 326 with non-diabetic ESRD. The original and replication analyses confirmed association with four SNPs in intron 13 (permutation p-values for combined analyses = 0.001-0.003), one in intron 1 (P=0.004) and one in intron 5 (P=0.002) with T2DM-associated ESRD. In a subsequent combined analysis of all 1,135 T2DM-ESRD cases and 1,160 controls, an additional 7 intron 13 SNPs produced evidence of association (P = 3.5×10-5 – P=0.05). No associations were seen with these SNPs in those with T2DM lacking nephropathy or with ESRD due to non-diabetic causes. Variants in intron 13 of the ELMO1 gene appear to confer risk for diabetic nephropathy in AA. PMID:19183347

Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice

PubMed Central

Badoni, Saurabh; Das, Sweta; Sayal, Yogesh K.; Gopalakrishnan, S.; Singh, Ashok K.; Rao, Atmakuri R.; Agarwal, Pinky; Parida, Swarup K.; Tyagi, Akhilesh K.

2016-01-01

We developed genome-wide 84634 ISM (intron-spanning marker) and 16510 InDel-fragment length polymorphism-based ILP (intron-length polymorphism) markers from genes physically mapped on 12 rice chromosomes. These genic markers revealed much higher amplification-efficiency (80%) and polymorphic-potential (66%) among rice accessions even by a cost-effective agarose gel-based assay. A wider level of functional molecular diversity (17–79%) and well-defined precise admixed genetic structure was assayed by 3052 genome-wide markers in a structured population of indica, japonica, aromatic and wild rice. Six major grain weight QTLs (11.9–21.6% phenotypic variation explained) were mapped on five rice chromosomes of a high-density (inter-marker distance: 0.98 cM) genetic linkage map (IR 64 x Sonasal) anchored with 2785 known/candidate gene-derived ISM and ILP markers. The designing of multiple ISM and ILP markers (2 to 4 markers/gene) in an individual gene will broaden the user-preference to select suitable primer combination for efficient assaying of functional allelic variation/diversity and realistic estimation of differential gene expression profiles among rice accessions. The genomic information generated in our study is made publicly accessible through a user-friendly web-resource, “Oryza ISM-ILP marker” database. The known/candidate gene-derived ISM and ILP markers can be enormously deployed to identify functionally relevant trait-associated molecular tags by optimal-resource expenses, leading towards genomics-assisted crop improvement in rice. PMID:27032371

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.

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...

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

Updated U.S. population standard for the Veterans RAND 12-item Health Survey (VR-12).

PubMed

Selim, Alfredo J; Rogers, William; Fleishman, John A; Qian, Shirley X; Fincke, Benjamin G; Rothendler, James A; Kazis, Lewis E

2009-02-01

The purpose of this project was to develop an updated U.S. population standard for the Veterans RAND 12-item Health Survey (VR-12). We used a well-defined and nationally representative sample of the U.S. population from 52,425 responses to the Medical Expenditure Panel Survey (MEPS) collected between 2000 and 2002. We applied modified regression estimates to update the non-proprietary 1990 scoring algorithms. We applied the updated standard to the Medicare Health Outcomes Survey (HOS) to compute the VR-12 physical (PCS((MEPS standard))) and mental (MCS((MEPS standard))) component summaries based on the MEPS. We compared these scores to PCS and MCS based on the 1990 U.S. population standard. Using the updated U.S. population standard, the average VR-12 PCS((MEPS standard)) and MCS((MEPS standard)) scores in the Medicare HOS were 39.82 (standard deviation [SD] = 12.2) and 50.08 (SD = 11.4), respectively. For the same Medicare HOS, the average PCS and MCS scores based on the 1990 standard were 1.40 points higher and 0.99 points lower in comparison to VR-12 PCS and MCS, respectively. Changes in the U.S. population between 1990 and today make the old standard obsolete for the VR-12, so the updated standard developed here is widely available to serve as such a contemporary standard for future applications for health-related quality of life (HRQoL) assessments.

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

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

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.

COL5A1: Fine genetic mapping, intron/exon organization, and exclusion as candidate gene in families with tuberous sclerosis complex 1, hereditary hemorrhagic telangiectasia, and Ehlers-Danlos syndrome type II

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

Greenspan, D.S.; Papenberg, K.A.; Marchuk, D.A.

1994-09-01

Type V collagen is the only fibrillar collagen which has yet to be implicated in the pathogenesis of genetic diseases in humans or mice. To begin examining the possible role of type V collagen in genetic disease, we have previously mapped COL5A1, the gene for the {alpha}1 chain of type V collagen, to 9q23.2{r_arrow}q34.3 and described two restriction site polymorphisms which allowed us to exclude COL5A1 as candidate gene for nail-patella syndrome. We have now used these polymorphisms to exclude COL5A1 as candidate gene for tuberous sclerosis complex 1 and Ehlers-Danlos syndrome type II. In addition, we describe a CAmore » repeat, with observed heterozygosity of about 0.5, in a COL5A1 intron, which has allowed us to exclude COL5A1 as a candidate gene in hereditary hemorrhagic telangiectasia and to place COL5A1 on the CEPH family genetic map between markers D9S66 and D9S67. We have also determined the entire intron/exon organization of COL5A1, which will facilitate characterization of mutations in genetic diseases with which COL5A1 may be linked in future studies.« less

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 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

Strong Signature of Natural Selection within an FHIT Intron Implicated in Prostate Cancer Risk

PubMed Central

Ding, Yan; Larson, Garrett; Rivas, Guillermo; Lundberg, Cathryn; Geller, Louis; Ouyang, Ching; Weitzel, Jeffrey; Archambeau, John; Slater, Jerry; Daly, Mary B.; Benson, Al B.; Kirkwood, John M.; O'Dwyer, Peter J.; Sutphen, Rebecca; Stewart, James A.; Johnson, David; Nordborg, Magnus; Krontiris, Theodore G.

2008-01-01

Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's χ2 = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's χ2 = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (π = 0.0072, Tajima's D = 3.31, 14 SNPs) and the Japanese (π = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer. PMID:18953408

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

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.

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

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

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

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

Deep intronic GPR143 mutation in a Japanese family with ocular albinism.

PubMed

Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

2015-06-10

Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease.

Sensing Self and Foreign Circular RNAs by Intron Identity.

PubMed

Chen, Y Grace; Kim, Myoungjoo V; Chen, Xingqi; Batista, Pedro J; Aoyama, Saeko; Wilusz, Jeremy E; Iwasaki, Akiko; Chang, Howard Y

2017-07-20

Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity. Copyright © 2017 Elsevier Inc. All rights reserved.

Nuclear introns outperform mitochondrial DNA in inter-specific phylogenetic reconstruction: Lessons from horseshoe bats (Rhinolophidae: Chiroptera).

PubMed

Dool, Serena E; Puechmaille, Sebastien J; Foley, Nicole M; Allegrini, Benjamin; Bastian, Anna; Mutumi, Gregory L; Maluleke, Tinyiko G; Odendaal, Lizelle J; Teeling, Emma C; Jacobs, David S

2016-04-01

DNA introgression. We demonstrated that by using just two introns one can recover a better supported species tree than when using the mtDNA alone, despite the shorter overall length of the combined introns. Additionally, when combining any single intron with mtDNA, we showed that the result is highly similar to the mtDNA gene tree and far from the true species tree and therefore this approach should be avoided. We caution against the indiscriminate use of mtDNA in phylogenetic studies and advocate for pilot studies to select nuclear introns. The selection of marker type and number is a crucial step that is best based on critical examination of preliminary or previously published data. Based on our findings and previous publications, we recommend the following markers to recover phylogenetic relationships between recently diverged taxa (<20 My) in bats and other mammals: ACOX2, COPS7A, BGN, ROGDI and STAT5A. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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

Is “Junk” DNA Mostly Intron DNA?

PubMed Central

Wong, Gane Ka-Shu; Passey, Douglas A.; Huang, Ying-zong; Yang, Zhiyong; Yu, Jun

2000-01-01

Among higher eukaryotes, very little of the genome codes for protein. What is in the rest of the genome, or the “junk” DNA, that, in Homo sapiens, is estimated to be almost 97% of the genome? Is it possible that much of this “junk” is intron DNA? This is not a question that can be answered just by looking at the published data, even from the finished genomes. One cannot assume that there are no genes in a sequenced region, just because no genes were annotated. We introduce another approach to this problem, based on an analysis of the cDNA-to-genomic alignments, in all of the complete or nearly-complete genomes from the multicellular organisms. Our conclusion is that, in animals but not in plants, most of the “junk” is intron DNA. PMID:11076852

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.

Intron retention in viruses and cellular genes: Detention, border controls and passports.

PubMed

Rekosh, David; Hammarskjold, Marie-Louise

2018-05-01

Intron retention (IR), where one or more introns remain in the RNA after splicing, was long thought to be rare in mammalian cells, albeit common in plants and some viruses. Largely due to the development of better methods for RNA analysis, it has now been recognized that IR is much more common than previously thought and that this mechanism is likely to play an important role in mammalian gene regulation. To date, most publications and reviews about IR have described the resulting mRNAs as "dead end" products, with no direct consequence for the proteome. However, there are also many reports of mRNAs with retained introns giving rise to alternative protein isoforms. Although this was originally revealed in viral systems, there are now numerous examples of bona fide cellular proteins that are translated from mRNAs with retained introns. These new isoforms have sometimes been shown to have important regulatory functions. In this review, we highlight recent developments in this area and the research on viruses that led the way to the realization of the many ways in which mRNAs with retained introns can be regulated. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA Export and Localization > Nuclear Export/Import RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes. © 2018 Wiley Periodicals, Inc.

15 CFR 801.12 - Rules and regulations for the BE-140, Benchmark Survey of Insurance Transactions by U.S...

Code of Federal Regulations, 2010 CFR

2010-01-01

... insurance company. Part 3 requests information needed to determine whether a report is required, the types..., Benchmark Survey of Insurance Transactions by U.S. Insurance Companies with Foreign Persons. 801.12 Section... Transactions by U.S. Insurance Companies with Foreign Persons. (a) The BE-140, Benchmark Survey of Insurance...

Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium

PubMed Central

Perrineau, Marie-Mathilde; Price, Dana C.; Mohr, Georg

2015-01-01

Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs) and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element) in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications. PMID:26157604

PPARA intron polymorphism associated with power performance in 30-s anaerobic Wingate Test.

PubMed

Petr, Miroslav; Stastny, Petr; Št'astný, Petr; Pecha, Ondřej; Šteffl, Michal; Šeda, Ondřej; Kohlíková, Eva

2014-01-01

To date, polymorphisms in several genes have been associated with a strength/power performance including alpha 3 actinin, ciliary neurotrophic factor, vitamin D receptor, or angiotensin I converting enzyme, underlining the importance of genetic component of the multifactorial strength/power-related phenotypes. The single nucleotide variation in peroxisome proliferator-activated receptor alpha gene (PPARA) intron 7 G/C (rs4253778; g.46630634G>C) has been repeatedly found to play a significant role in response to different types of physical activity. We investigated the effect of PPARA intron 7 G/C polymorphism specifically on anaerobic power output in a group of 77 elite male Czech ice hockey players (18-36 y). We determined the relative peak power per body weight (Pmax.kg(-1)) and relative peak power per fat free mass (W.kg(-1)FFM) during the 30-second Wingate Test (WT30) on bicycle ergometer (Monark 894E Peak bike, MONARK, Sweden). All WT30s were performed during the hockey season. Overall genotype frequencies were 50.6% GG homozygotes, 40.3% CG heterozygotes, and 9.1% CC homozygotes. We found statistically significant differences in Pmax.kg(-1) and marginally significant differences in Pmax.kg(-1)FFM values in WT30 between carriers and non-carriers for C allele (14.6 ± 0.2 vs. 13.9 ± 0.3 W.kg(-1) and 15.8 ± 0.2 vs. 15.2 ± 0.3 W.kg(-1)FFM, P = 0.036 and 0.12, respectively). Furthermore, Pmax.kg(-1)FFM strongly positively correlated with the body weight only in individuals with GG genotypes (R = 0.55; p<0.001). Our results indicate that PPARA 7C carriers exhibited higher speed strength measures in WT30. We hypothesize that C allele carriers within the cohort of trained individuals may possess a metabolic advantage towards anaerobic metabolism.

Deep intronic GPR143 mutation in a Japanese family with ocular albinism

PubMed Central

Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

2015-01-01

Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease. PMID:26061757

Functional understanding of the diverse exon-intron structures of human GPCR genes.

PubMed

Hammond, Dorothy A; Olman, Victor; Xu, Ying

2014-02-01

The GPCR genes have a variety of exon-intron structures even though their proteins are all structurally homologous. We have examined all human GPCR genes with at least two functional protein isoforms, totaling 199, aiming to gain an understanding of what may have contributed to the large diversity of the exon-intron structures of the GPCR genes. The 199 genes have a total of 808 known protein splicing isoforms with experimentally verified functions. Our analysis reveals that 1301 (80.6%) adjacent exon-exon pairs out of the total of 1,613 in the 199 genes have either exactly one exon skipped or the intron in-between retained in at least one of the 808 protein splicing isoforms. This observation has a statistical significance p-value of 2.051762 * e(-09), assuming that the observed splicing isoforms are independent of the exon-intron structures. Our interpretation of this observation is that the exon boundaries of the GPCR genes are not randomly determined; instead they may be selected to facilitate specific alternative splicing for functional purposes.

11 CFR 100.11 - State (2 U.S.C. 431(12)).

Code of Federal Regulations, 2010 CFR

2010-01-01

... 11 Federal Elections 1 2010-01-01 2010-01-01 false State (2 U.S.C. 431(12)). 100.11 Section 100.11 Federal Elections FEDERAL ELECTION COMMISSION GENERAL SCOPE AND DEFINITIONS (2 U.S.C. 431) General Definitions § 100.11 State (2 U.S.C. 431(12)). State means each State of the United States, the District of...

TSHR intronic polymorphisms (rs179247 and rs12885526) and their role in the susceptibility of the Brazilian population to Graves' disease and Graves' ophthalmopathy.

PubMed

Bufalo, N E; Dos Santos, R B; Marcello, M A; Piai, R P; Secolin, R; Romaldini, J H; Ward, L S

2015-05-01

Intronic thyroid-stimulating hormone receptor polymorphisms have been associated with the risk for both Graves' disease and Graves' ophthalmopathy, but results have been inconsistent among different populations. We aimed to investigate the influence of thyroid-stimulating hormone receptor intronic polymorphisms in a large well-characterized population of GD patients. We studied 279 Graves' disease patients (231 females and 48 males, 39.80 ± 11.69 years old), including 144 with Graves' ophthalmopathy, matched to 296 healthy control individuals. Thyroid-stimulating hormone receptor genotypes of rs179247 and rs12885526 were determined by Real Time PCR TaqMan(®) SNP Genotyping. A multivariate analysis showed that the inheritance of the thyroid-stimulating hormone receptor AA genotype for rs179247 increased the risk for Graves' disease (OR = 2.821; 95 % CI 1.595-4.990; p = 0.0004), whereas the thyroid-stimulating hormone receptor GG genotype for rs12885526 increased the risk for Graves' ophthalmopathy (OR = 2.940; 95 % CI 1.320-6.548; p = 0.0083). Individuals with Graves' ophthalmopathy also presented lower mean thyrotropin receptor antibodies levels (96.3 ± 143.9 U/L) than individuals without Graves' ophthalmopathy (98.3 ± 201.9 U/L). We did not find any association between the investigated polymorphisms and patients clinical features or outcome. We demonstrate that thyroid-stimulating hormone receptor intronic polymorphisms are associated with the susceptibility to Graves' disease and Graves' ophthalmopathy in the Brazilian population, but do not appear to influence the disease course.

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

PubMed Central

Hotto, Amber M.; Castandet, Benoît; Gilet, Laetitia; Higdon, Andrea; Condon, Ciarán; Stern, David B.

2015-01-01

RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3′ end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3′ extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation. PMID:25724636

Limited MHC class I intron 2 repertoire variation in bonobos.

PubMed

de Groot, Natasja G; Heijmans, Corrine M C; Helsen, Philippe; Otting, Nel; Pereboom, Zjef; Stevens, Jeroen M G; Bontrop, Ronald E

2017-10-01

Common chimpanzees (Pan troglodytes) experienced a selective sweep, probably caused by a SIV-like virus, which targeted their MHC class I repertoire. Based on MHC class I intron 2 data analyses, this selective sweep took place about 2-3 million years ago. As a consequence, common chimpanzees have a skewed MHC class I repertoire that is enriched for allotypes that are able to recognise conserved regions of the SIV proteome. The bonobo (Pan paniscus) shared an ancestor with common chimpanzees approximately 1.5 to 2 million years ago. To investigate whether the signature of this selective sweep is also detectable in bonobos, the MHC class I gene repertoire of two bonobo panels comprising in total 29 animals was investigated by Sanger sequencing. We identified 14 Papa-A, 20 Papa-B and 11 Papa-C alleles, of which eight, five and eight alleles, respectively, have not been reported previously. Within this pool of MHC class I variation, we recovered only 2 Papa-A, 3 Papa-B and 6 Papa-C intron 2 sequences. As compared to humans, bonobos appear to have an even more diminished MHC class I intron 2 lineage repertoire than common chimpanzees. This supports the notion that the selective sweep may have predated the speciation of common chimpanzees and bonobos. The further reduction of the MHC class I intron 2 lineage repertoire observed in bonobos as compared to the common chimpanzee may be explained by a founding effect or other subsequent selective processes.

A few nucleotide polymorphisms are sufficient to recruit nuclear factors differentially to the intron 1 of HPV-16 intratypic variants.

PubMed

López-Urrutia, Eduardo; Valdés, Jesús; Bonilla-Moreno, Raúl; Martínez-Salazar, Martha; Martínez-Garcia, Martha; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2012-06-01

The HPV-16 E6/E7 genes, which contain intron 1, are processed by alternative splicing and its transcripts are detected with a heterogeneous profile in tumours cells. Frequently, the HPV-16 positive carcinoma cells bear viral variants that contain single nucleotide polymorphisms into its DNA sequence. We were interested in analysing the contribution of this polymorphism to the heterogeneity in the pattern of the E6/E7 spliced transcripts. Using the E6/E7 sequences from three closely related HPV-16 variants, we have shown that a few nucleotide changes are sufficient to produce heterogeneity in the splicing profile. Furthermore, using mutants that contained a single SNP, we also showed that one nucleotide change was sufficient to reproduce the heterogeneous splicing profile. Additionally, a difference of two or three SNPs among these viral sequences was sufficient to recruit differentially several splicing factors to the polymorphic E6/E7 transcripts. Moreover, only one SNP was sufficient to alter the binding site of at least one splicing factor, changing the ability of splicing factors to bind the transcript. Finally, the factors that were differentially bound to the short form of intron 1 of one of these E6/E7 variants were identified as TIA1 and/or TIAR and U1-70k, while U2AF65, U5-52k and PTB were preferentially bound to the transcript of the other variants. Copyright © 2012 Elsevier B.V. All rights reserved.

Bacterial Group II Introns: Identification and Mobility Assay.

PubMed

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

2016-01-01

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

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.

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.

Geological Survey investigations in the U12e.05 tunnel, Nevada Test Site

USGS Publications Warehouse

Diment, William H.; Wilmarth, V.R.; Houser, F.N.; Dickey, D.D.; Hinrichs, E.N.; Botinelly, T.; Wilcox, R.E.; Byers, F.M.

1959-01-01

The U12e.05 tunnel, one of two laterals from the main U12e tunnel, trends west and connects with the main tunnel about U960 feet from the portal (fig. 2). The U12e.05 tunnel was driven for the nuclear test, code name Blanca, which took place on October 30, 1958. Before the explosion, the tunnel was 8 feet high and 8 to 9 feet wide and consisted of 990 feet of workings, a shot chamber, and an alcove (fig. 2). The original shot chamber, at the west end of the tunnel, was 19 feet long, 10 feet wide, and 15 feet high. The vertical and minimum cover over the original shot chamber are 1,150 and 950 feet, respectively. After detonation of the nuclear device in the test, code named Logan, in the U12e.02 tunnel on October 15, 1958, the U12e.05 tunnel, locally, was damaged severely.

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

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.

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 editing in the anticodon of tRNA Leu (CAA) occurs before group I intron splicing in plastids of a moss Takakia lepidozioides S. Hatt. & Inoue.

PubMed

Miyata, Y; Sugita, C; Maruyama, K; Sugita, M

2008-03-01

RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of most land plants. In this study, we amplified and sequenced the group I intron-containing tRNA Leu gene, trnL-CAA, from Takakia lepidozioides, a moss. DNA sequence analysis revealed that the T. lepidozioides tRNA Leu gene consisted of a 35-bp 5' exon, a 469-bp group I intron and a 50-bp 3' exon. The intron was inserted between the first and second position of the tRNA Leu anticodon. In general, plastid tRNA Leu genes with a group I intron code for a TAA anticodon in most land plants. This strongly suggests that the first nucleotide of the CAA anticodon could be edited in T. lepidozioides plastids. To investigate this possibility, we analysed cDNAs derived from the trnL-CAA transcripts. We demonstrated that the first nucleotide C of the anticodon was edited to create a canonical UAA anticodon in T. lepidozioides plastids. cDNA sequencing analyses of the spliced or unspliced tRNA Leu transcripts revealed that, while the spliced tRNA was completely edited, editing in the unspliced tRNAs were only partial. This is the first experimental evidence that the anticodon editing of tRNA occurs before RNA splicing in plastids. This suggests that this editing is a prerequisite to splicing of pre-tRNA Leu.

Alternative intronic promoters in development and disease.

PubMed

Vacik, Tomas; Raska, Ivan

2017-05-01

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.

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.

DIVERSITY OF THE TYPE 1 INTRON-ITS REGION OF THE 18S rRNA GENE IN PSEUDOGYMNOASCUS SPECIES FROM THE RED HILLS OF KANSAS.

PubMed

Chen, Xi; Crupper, Scott S

2016-09-01

Gypsum caves found throughout the Red Hills of Kansas have the state's most diverse and largest population of cave-roosting bats. White-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans, which threatens all temperate bat species, has not been previously detected in the gypsum caves as this disease moves westward from the eastern United States. Cave soil was obtained from the gypsum caves, and using the polymerase chain reaction, a 624-nucleotide DNA fragment specific to the Type 1 intron-internal transcribed spacer region of the 18S rRNA gene from Pseudogymnoascus species was amplified. Subsequent cloning and DNA sequencing indicated P. destructans DNA was present, along with 26 uncharacterized Pseudogymnoascus DNA variants. However, no evidence of WNS was observed in bat populations residing in these caves.

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.

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

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

Interleukin-4 Gene Intron 3 VNTR Polymorphism in Type 2 Diabetes Patients with Peripheral Neuropathy.

PubMed

Buraczynska, Monika; Buraczynska, Kinga; Zukowski, Pawel; Ksiazek, Andrzej

2018-02-01

Diabetic peripheral neuropathy (DPN) is one of late complications of diabetes mellitus. The aim of this study was to evaluate the association between variable number tandem repeat (VNTR) polymorphism in intron 3 of interleukin-4 gene and risk of DPN. We examined 926 T2DM patients and 420 healthy controls. In the patient group, 44% had DPN. Genomic DNA was isolated from all subjects and genotyped for the IL-4 VNTR polymorphism by polymerase chain reaction (PCR). No significant difference was observed in the frequency of minor P1 allele between T2DM patients and controls (OR 1.00, 95% CI 0.81-1.23, p = 0.988). The distribution of IL-4 VNTR polymorphism was compared between patients with DPN and those without it. The polymorphism was not significantly associated with DPN in studied subjects. In comparison of 406 T2DM patients with DPN and 520 patients without it, the OR (95% CI) for P1 allele was 0.82 (0.65-1.04), p = 0.10 and for P1P1 genotype 1.00 (0.53-1.89), p = 0.991. When two subgroups of patients with DPN, those with cardiovascular disease (CVD) and without CVD, were compared, subgroup with coexisting CVD had significantly higher frequency of P1 allele than patients without CVD, with odds ratio for the P1 allele 3.27 (95% CI 1.83-5.83), p = 0.0001. Our results demonstrated that VNTR polymorphism in the IL-4 gene is associated with DPN in type 2 diabetes patients with coexisting CVD.

Genome-wide identification of conserved intronic non-coding sequences using a Bayesian segmentation approach.

PubMed

Algama, Manjula; Tasker, Edward; Williams, Caitlin; Parslow, Adam C; Bryson-Richardson, Robert J; Keith, Jonathan M

2017-03-27

Computational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences. We identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors. This study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed. Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome

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

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.

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

Intron size and genome size in plants.

Treesearch

J. Wendel; R. Cronn; I. Alvarez; B. Liu; R. Small; D. Senchina

2002-01-01

It has long been known that genomes vary over a remarkable range of sizes in both plants (Bennett, Cox, and Leitch 1997) and animals (Gregory 2001). It also has become evident that across the broad phylogenetic sweep, genome size may be correlated with intron size (Deutsch and Long 1999; Vinogradov 1999; McLysaght et al. 2000), suggesting that some component of genome...

Sustained glycaemic control and less nocturnal hypoglycaemia with insulin glargine 300U/mL compared with glargine 100U/mL in Japanese adults with type 1 diabetes (EDITION JP 1 randomised 12-month trial including 6-month extension).

PubMed

Matsuhisa, Munehide; Koyama, Masayoshi; Cheng, Xi; Sumi, Mariko; Riddle, Matthew C; Bolli, Geremia B; Hirose, Takahisa

2016-12-01

To evaluate the efficacy and safety of insulin glargine 300U/mL (Gla-300) versus glargine 100U/mL (Gla-100) in adults with type 1 diabetes in Japan over 12months. EDITION JP 1 was a multicentre, randomised, open-label phase 3 study. Following a 6-month on-treatment period, participants continued to receive Gla-300 or Gla-100 once daily, plus mealtime insulin, over a 6-month open-label extension phase. HbA1c, hypoglycaemia, body weight and adverse events were assessed. Overall, 114/122 (93%) and 114/121 (94%) of participants in the Gla-300 and Gla-100 group, respectively, completed the 6-month extension phase. Glycaemic control was sustained in both groups up to month 12 (mean HbA1c: Gla-300, 7.9% [62mmol/mol]; Gla-100, 7.8% [62mmol/mol]). Annualised rates of hypoglycaemia were lower with Gla-300 versus Gla-100; significantly for nocturnal confirmed (<3.0mmol/L [<54mg/dL]) or severe hypoglycaemia (2.39 and 3.85 events per participant-year; rate ratio: 0.62 [0.39-0.97]). No between-treatment differences in mean body weight change or adverse events were observed. Over 12months' treatment, participants with type 1 diabetes receiving Gla-300 achieved sustained glycaemic control and experienced less nocturnal hypoglycaemia that was confirmed (<3.0mmol/L [<54mg/dL]) or severe compared with Gla-100, supporting the 6-month results. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

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

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.

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.

Estimation of mercury amount in the components of spent U-type lamp.

PubMed

Rhee, Seung-Whee

2017-05-01

Spent U-type lamps are strongly encouraged to be separately managed in Korea, because U-type lamps are categorized as a household waste and thereby could not be managed properly. Determination of mercury amount in the components of U-type lamp, such as plastics, glass tube and phosphor powder from 3 U-type lamp manufacturers (A, B and C), is carried out to estimate the mercury content in spent U-type lamps. Regardless of lamp manufacturers, the portion of mercury in phosphor powder was higher than 90%, but that in plastics and others was less than 1%. At an air flow rate of 1.0 L/min, the range of the initial mercury concentration in vapor phase for U-type lamp was between 849 and 2076 µg/m 3 from 3 companies. The estimated mercury amount in vapor phase of U-type lamp was in the range from 0.206 mg for company A to 0.593 mg for company B. And the portion of mercury in vapor phase in the total amount of mercury was estimated in the range from 3.0% for company A to 6.7% for company B. Hence, it is desirable to get rid of mercury from phosphor powder in order to perform U-type lamps recycling.

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

PubMed

Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

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

Gene encoding the human. beta. -hexosaminidase. beta. chain: Extensive homology of intron placement in the. alpha. - and. beta. -chain genes

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

Proia, R.L.

1988-03-01

Lysosomal {beta}-hexosaminidase is composed of two structurally similar chains, {alpha} and {beta}, that are the products of different genes. Mutations in either gene causing {beta}-hexosaminidase deficiency result in the lysosomal storage disease GM2-gangliosidosis. To enable the investigation of the molecular lesions in this disorder and to study the evolutionary relationship between the {alpha} and {beta} chains, the {beta}-chain gene was isolated, and its organization was characterized. The {beta}-chain coding region is divided into 14 exons distributed over {approx}40 kilobases of DNA. Comparison with the {alpha}-chain gene revealed that 12 of the 13 introns interrupt the coding regions at homologous positions.more » This extensive sharing of intron placement demonstrates that the {alpha} and {beta} chains evolved by way of the duplication of a common ancestor.« less

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

PubMed Central

Bornstein, P; McKay, J; Liska, D J; Apone, S; Devarayalu, S

1988-01-01

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control. Images PMID:3211130

12 CFR 7.5009 - Location under 12 U.S.C. 85 of national banks operating exclusively through the Internet.

Code of Federal Regulations, 2012 CFR

2012-01-01

... operating exclusively through the Internet. 7.5009 Section 7.5009 Banks and Banking COMPTROLLER OF THE... under 12 U.S.C. 85 of national banks operating exclusively through the Internet. For purposes of 12 U.S.C. 85, the main office of a national bank that operates exclusively through the Internet is the...

12 CFR 7.5009 - Location under 12 U.S.C. 85 of national banks operating exclusively through the Internet.

Code of Federal Regulations, 2013 CFR

2013-01-01

... operating exclusively through the Internet. 7.5009 Section 7.5009 Banks and Banking COMPTROLLER OF THE... under 12 U.S.C. 85 of national banks operating exclusively through the Internet. For purposes of 12 U.S.C. 85, the main office of a national bank that operates exclusively through the Internet is the...

12 CFR 7.5009 - Location under 12 U.S.C. 85 of national banks operating exclusively through the Internet.

Code of Federal Regulations, 2014 CFR

2014-01-01

... operating exclusively through the Internet. 7.5009 Section 7.5009 Banks and Banking COMPTROLLER OF THE... under 12 U.S.C. 85 of national banks operating exclusively through the Internet. For purposes of 12 U.S.C. 85, the main office of a national bank that operates exclusively through the Internet is the...

12 CFR 7.5009 - Location under 12 U.S.C. 85 of national banks operating exclusively through the Internet.

Code of Federal Regulations, 2011 CFR

2011-01-01

... operating exclusively through the Internet. 7.5009 Section 7.5009 Banks and Banking COMPTROLLER OF THE... under 12 U.S.C. 85 of national banks operating exclusively through the Internet. For purposes of 12 U.S.C. 85, the main office of a national bank that operates exclusively through the Internet is the...

12 CFR 7.5009 - Location under 12 U.S.C. 85 of national banks operating exclusively through the Internet.

Code of Federal Regulations, 2010 CFR

2010-01-01

... operating exclusively through the Internet. 7.5009 Section 7.5009 Banks and Banking COMPTROLLER OF THE... under 12 U.S.C. 85 of national banks operating exclusively through the Internet. For purposes of 12 U.S.C. 85, the main office of a national bank that operates exclusively through the Internet is the...

12 CFR 5.63 - Capital limitation under 12 U.S.C. 56.

Code of Federal Regulations, 2011 CFR

2011-01-01

... permanent capital. Further, a national bank may not declare a dividend in excess of undivided profits. (b... the undivided profits of the national bank are not sufficient to cover a proposed dividend on preferred stock, the proposed dividend constitutes a reduction in capital subject to 12 U.S.C. 59 and § 5.46. ...

12 CFR 5.63 - Capital limitation under 12 U.S.C. 56.

Code of Federal Regulations, 2010 CFR

2010-01-01

... permanent capital. Further, a national bank may not declare a dividend in excess of undivided profits. (b... the undivided profits of the national bank are not sufficient to cover a proposed dividend on preferred stock, the proposed dividend constitutes a reduction in capital subject to 12 U.S.C. 59 and § 5.46. ...

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

U.S./U.S.S.R. SYMPOSIUM ON PARTICULATE CONTROL (3RD) HELD AT SUZDAL, U.S.S.R. ON SEPTEMBER 10-12, 1979

EPA Science Inventory

The proceedings document the Third U.S./U.S.S.R. Symposium on Particulate Control, September 10-12, 1979, in Suzdal, U.S.S.R. Papers covered such topics as: predicting back-corona formation and fly ash resistivity, improved electrostatic precipitator (ESP) mathematical modeling, ...

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.

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

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.

Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients.

PubMed

Petersen, Sanne M; Dandanell, Mette; Rasmussen, Lene J; Gerdes, Anne-Marie; Krogh, Lotte N; Bernstein, Inge; Okkels, Henrik; Wikman, Friedrik; Nielsen, Finn C; Hansen, Thomas V O

2013-10-03

Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance. Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing. We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A > T, MLH1 c.1039-8 T > A, MSH2 c.2459-18delT, and MSH6 c.3439-16C > T). In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.

Cantilever-type Thermal Microactuators Fabricated by SOI-MUMPs with U-type and I-type Configurations

NASA Astrophysics Data System (ADS)

Osada, Takahiro; Ochiai, Kuniyuki; Osada, Kazuki; Muro, Hideo

Recently, the micro fluid systems have been extensively studied, where microactuators such as micro valves fabricated by MEMS technology are essential for realizing these systems. In this paper thermal microactuators with U-type and I-type shapes fabricated by SOI-MUMPs technology have been investigated for optimizing their configurations.

Evolution of the tRNALeu (UAA) Intron and Congruence of Genetic Markers in Lichen-Symbiotic Nostoc

PubMed Central

Kaasalainen, Ulla; Olsson, Sanna; Rikkinen, Jouko

2015-01-01

The group I intron interrupting the tRNALeu UAA gene (trnL) is present in most cyanobacterial genomes as well as in the plastids of many eukaryotic algae and all green plants. In lichen symbiotic Nostoc, the P6b stem-loop of trnL intron always involves one of two different repeat motifs, either Class I or Class II, both with unresolved evolutionary histories. Here we attempt to resolve the complex evolution of the two different trnL P6b region types. Our analysis indicates that the Class II repeat motif most likely appeared first and that independent and unidirectional shifts to the Class I motif have since taken place repeatedly. In addition, we compare our results with those obtained with other genetic markers and find strong evidence of recombination in the 16S rRNA gene, a marker widely used in phylogenetic studies on Bacteria. The congruence of the different genetic markers is successfully evaluated with the recently published software Saguaro, which has not previously been utilized in comparable studies. PMID:26098760

Evolution of the tRNALeu (UAA) Intron and Congruence of Genetic Markers in Lichen-Symbiotic Nostoc.

PubMed

Kaasalainen, Ulla; Olsson, Sanna; Rikkinen, Jouko

2015-01-01

The group I intron interrupting the tRNALeu UAA gene (trnL) is present in most cyanobacterial genomes as well as in the plastids of many eukaryotic algae and all green plants. In lichen symbiotic Nostoc, the P6b stem-loop of trnL intron always involves one of two different repeat motifs, either Class I or Class II, both with unresolved evolutionary histories. Here we attempt to resolve the complex evolution of the two different trnL P6b region types. Our analysis indicates that the Class II repeat motif most likely appeared first and that independent and unidirectional shifts to the Class I motif have since taken place repeatedly. In addition, we compare our results with those obtained with other genetic markers and find strong evidence of recombination in the 16S rRNA gene, a marker widely used in phylogenetic studies on Bacteria. The congruence of the different genetic markers is successfully evaluated with the recently published software Saguaro, which has not previously been utilized in comparable studies.

A novel non-coding RNA within an intron of CDH2 and association of its SNP with non-syndromic cleft lip and palate.

PubMed

Kumari, Priyanka; Singh, Subodh Kumar; Raman, Rajiva

2018-06-05

Genome-wide linkage analysis and whole genome sequencing in a Van der Woude syndrome (VWS) family revealed that the SNP, rs539075, within intron 2 of the cadherin 2 gene (CDH2) co-segregated with the disease phenotype. A study with nonsyndromic cleft lip with or without cleft palate (NSCL ± P) cases (N = 292) and controls (N = 287) established association of this SNP with NSCL ± P as a risk factor. RT-PCR based expression analysis of the SNP-harbouring region of intron 2 of CDH2 in the clefted lip and/or palate tissues of 16 patients revealed that the mutant allele expressed in all those individuals having it (hetero-/homozygous), whereas the wild type allele expressed in <50% of the samples in which it was present. The intronic transcript was also present in the prospective lip and palate region of 13.5 dpc mouse embryo, detected by RNA in situ hybridization and RT-PCR. These results including the in silico, characterization of the ~200 nt-intronic transcript showed that conformationally it fits best with noncoding small RNA, possibly a precursor of miRNA. Its function in the orofacial organogenesis remains to be elucidated which will enable us to define the role of this mutant ncRNA in the clefting of lip and palate. Copyright © 2018 Elsevier B.V. All rights reserved.

Intriguing Balancing Selection on the Intron 5 Region of LMBR1 in Human Population

PubMed Central

He, Fang; Wu, Dong-Dong; Kong, Qing-Peng; Zhang, Ya-Ping

2008-01-01

Background The intron 5 of gene LMBR1 is the cis-acting regulatory module for the sonic hedgehog (SHH) gene. Mutation in this non-coding region is associated with preaxial polydactyly, and may play crucial roles in the evolution of limb and skeletal system. Methodology/Principal Findings We sequenced a region of the LMBR1 gene intron 5 in East Asian human population, and found a significant deviation of Tajima's D statistics from neutrality taking human population growth into account. Data from HapMap also demonstrated extended linkage disequilibrium in the region in East Asian and European population, and significantly low degree of genetic differentiation among human populations. Conclusion/Significance We proposed that the intron 5 of LMBR1 was presumably subject to balancing selection during the evolution of modern human. PMID:18698406

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.

Platelet monoamine oxidase type B, MAOB intron 13 and MAOA-uVNTR polymorphism and symptoms of post-traumatic stress disorder.

PubMed

Svob Strac, Dubravka; Kovacic Petrovic, Zrnka; Nikolac Perkovic, Matea; Umolac, Danica; Nedic Erjavec, Gordana; Pivac, Nela

2016-07-01

Post-traumatic stress disorder (PTSD), a disorder that develops following exposure to traumatic experience(s), is frequently associated with agitation, aggressive behavior and psychotic symptoms. Monoamine oxidase (MAO) degrades different biogenic amines and regulates mood, emotions and behavior, and has a role in the pathophysiology of various neuropsychiatric disorders. The aim of the study was to investigate the association between different symptoms occurring in PTSD [PTSD symptom severity assessed by the Clinician Administered PTSD Scale (CAPS), agitation and selected psychotic symptoms assessed by the Positive and Negative Syndrome Scale (PANSS)] and platelet MAO-B activity and/or genetic variants of MAOB rs1799836 and MAOA-uVNTR polymorphisms in 249 Croatian male veterans with PTSD. Our study revealed slightly higher platelet MAO-B activity in veterans with PTSD with more severe PTSD symptoms and in veterans with agitation, and significantly higher platelet MAO-B activity in veterans with more pronounced psychotic symptoms compared to veterans with less pronounced psychotic symptoms. Platelet MAO-B activity was associated with smoking but not with age. Genetic variants of MAOB rs1799836 and MAOA-uVNTR were not associated with agitation and selected psychotic symptoms in veterans with PTSD. A marginally significant association was found between MAOB rs1799836 polymorphism and severity of PTSD symptoms, but it was not confirmed since carriers of G or A allele of MAOB rs1799836 did not differ in their total CAPS scores. These findings suggest an association of platelet MAO-B activity, but a lack of association of MAOB rs1799836 and MAOA-uVNTR, with selected psychotic symptoms in ethnically homogenous veterans with PTSD.

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.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

PubMed

Chiu, Yung-Tuen; Wong, John K L; Choi, Shing-Wan; Sze, Karen M F; Ho, Daniel W H; Chan, Lo-Kong; Lee, Joyce M F; Man, Kwan; Cherny, Stacey; Yang, Wan-Ling; Wong, Chun-Ming; Sham, Pak-Chung; Ng, Irene O L

2016-06-01

Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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.

The intron 1 of HPV 16 has a suboptimal branch point at a guanosine.

PubMed

De la Rosa-Rios, Marco Antonio; Martínez-Salazar, Martha; Martínez-Garcia, Martha; González-Bonilla, César; Villegas-Sepúlveda, Nicolás

2006-06-01

The branch point sequence (BPS) of intron 1 of the HPV-16 was determined via RT-PCR in a cell free system, using lariat intermediates obtained by in vitro splicing reactions. We used synthetic E6/E7 transcripts and HeLa nuclear protein extracts to obtain the splicing intermediates. Then, a divergent oligonucleotide primer set, pairing on the lariat RNA that encompassed the 2'-5' phosphodiester bond formed between the 5' end of the intron and the BPS, was used for cDNA synthesis and PCR amplification. Subsequent RT-PCR assays revealed four splicing intermediates, made up of a major intermediary corresponding to the BPS and four cryptic branched sequences. Only intermediates bound at the 5' end of the intron are probably the authentic branch point sequence, and all of them branch at guanosine 328 instead of the typical adenosine. Unusually, the BPS of intron 1 of HPV-16 is a suboptimal sequence (AGUGAGU) that differs from the eukaryotic consensus BPS, which correlates with the splicing profile observed for early transcripts of HPV-16 in tumors and tumor derived cell lines. The implications of this unusual branch point sequence for splicing of the HPV-16 pre-mRNA are discussed.

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

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

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.

A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis

DOE PAGES

Pimentel, Harold; Parra, Marilyn; Gee, Sherry L.; ...

2015-11-03

Differentiating erythroblasts execute a dynamic alternative splicing program shown here to include extensive and diverse intron retention (IR) events. Cluster analysis revealed hundreds of developmentallydynamic introns that exhibit increased IR in mature erythroblasts, and are enriched in functions related to RNA processing such as SF3B1 spliceosomal factor. Distinct, developmentally-stable IR clusters are enriched in metal-ion binding functions and include mitoferrin genes SLC25A37 and SLC25A28 that are critical for iron homeostasis. Some IR transcripts are abundant, e.g. comprising ~50% of highly-expressed SLC25A37 and SF3B1 transcripts in late erythroblasts, and thereby limiting functional mRNA levels. IR transcripts tested were predominantly nuclearlocalized. Splicemore » site strength correlated with IR among stable but not dynamic intron clusters, indicating distinct regulation of dynamically-increased IR in late erythroblasts. Retained introns were preferentially associated with alternative exons with premature termination codons (PTCs). High IR was observed in disease-causing genes including SF3B1 and the RNA binding protein FUS. Comparative studies demonstrated that the intron retention program in erythroblasts shares features with other tissues but ultimately is unique to erythropoiesis. Finally, we conclude that IR is a multi-dimensional set of processes that post-transcriptionally regulate diverse gene groups during normal erythropoiesis, misregulation of which could be responsible for human disease.« less

A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis

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

Pimentel, Harold; Parra, Marilyn; Gee, Sherry L.

Differentiating erythroblasts execute a dynamic alternative splicing program shown here to include extensive and diverse intron retention (IR) events. Cluster analysis revealed hundreds of developmentallydynamic introns that exhibit increased IR in mature erythroblasts, and are enriched in functions related to RNA processing such as SF3B1 spliceosomal factor. Distinct, developmentally-stable IR clusters are enriched in metal-ion binding functions and include mitoferrin genes SLC25A37 and SLC25A28 that are critical for iron homeostasis. Some IR transcripts are abundant, e.g. comprising ~50% of highly-expressed SLC25A37 and SF3B1 transcripts in late erythroblasts, and thereby limiting functional mRNA levels. IR transcripts tested were predominantly nuclearlocalized. Splicemore » site strength correlated with IR among stable but not dynamic intron clusters, indicating distinct regulation of dynamically-increased IR in late erythroblasts. Retained introns were preferentially associated with alternative exons with premature termination codons (PTCs). High IR was observed in disease-causing genes including SF3B1 and the RNA binding protein FUS. Comparative studies demonstrated that the intron retention program in erythroblasts shares features with other tissues but ultimately is unique to erythropoiesis. Finally, we conclude that IR is a multi-dimensional set of processes that post-transcriptionally regulate diverse gene groups during normal erythropoiesis, misregulation of which could be responsible for human disease.« less

The Peculiar Characteristics of Fish Type I Interferons

PubMed Central

Boudinot, Pierre; Langevin, Christelle; Secombes, Christopher J.; Levraud, Jean-Pierre

2016-01-01

Antiviral type I interferons (IFNs) have been discovered in fish. Genomic studies revealed their considerable number in many species; some genes encode secreted and non-secreted isoforms. Based on cysteine motifs, fish type I IFNs fall in two subgroups, which use two different receptors. Mammalian type I IFN genes are intronless while type III have introns; in fish, all have introns, but structurally, both subgroups belong to type I. Type I IFNs likely appeared early in vertebrates as intron containing genes, and evolved in parallel in tetrapods and fishes. The diversity of their repertoires in fish and mammals is likely a convergent feature, selected as a response to the variety of viral strategies. Several alternative nomenclatures have been established for different taxonomic fish groups, calling for a unified system. The specific functions of each type I gene remains poorly understood, as well as their interactions in antiviral responses. However, distinct induction pathways, kinetics of response, and tissue specificity indicate that fish type I likely are highly specialized, especially in groups where they are numerous such as salmonids or cyprinids. Unravelling their functional integration constitutes the next challenge to understand how these cytokines evolved to orchestrate antiviral innate immunity in vertebrates. PMID:27827855

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.

An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation.

PubMed

Naro, Chiara; Jolly, Ariane; Di Persio, Sara; Bielli, Pamela; Setterblad, Niclas; Alberdi, Antonio J; Vicini, Elena; Geremia, Raffaele; De la Grange, Pierre; Sette, Claudio

2017-04-10

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species.

PubMed

Liu, Yanbin; Yap, Sihui Amy; Koh, Chong Mei John; Ji, Lianghui

2016-11-25

Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts. The upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4-11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase β subunit gene (FAS1) exhibited intermediate strength, displaying 50-160 and 20-90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6-3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5-3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation. Intronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are

A mutation in yeast mitochondrial DNA results in a precise excision of the terminal intron of the cytochrome b gene.

PubMed

Hill, J; McGraw, P; Tzagoloff, A

1985-03-25

The yeast nuclear gene CBP2 was previously proposed to code for a protein necessary for processing of the terminal intron in the cytochrome b pre-mRNA (McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468). In the present study we describe a mitochondrial mutation capable of suppressing the respiratory deficiency of cbp2 mutants. The mitochondrial suppressor mutation has been shown to be the result of a precise excision of the last intervening sequence from the cytochrome b gene. Strains with the altered mitochondrial DNA have normal levels of mature cytochrome b mRNA and of cytochrome b and exhibit wild type growth on glycerol. These results confirm that CBP2 codes for a protein specifically required for splicing of the cytochrome b intron and further suggest that absence of the intervening sequence does not noticeably affect the expression of respiratory function in mitochondria.

32 CFR 536.12 - Commanding General, U.S. Army Medical Command.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 3 2011-07-01 2009-07-01 true Commanding General, U.S. Army Medical Command... AND ACCOUNTS CLAIMS AGAINST THE UNITED STATES The Army Claims System § 536.12 Commanding General, U.S. Army Medical Command. (a) After consulting with the Commander USARCS on the selection of medical claims...

32 CFR 536.12 - Commanding General, U.S. Army Medical Command.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 3 2012-07-01 2009-07-01 true Commanding General, U.S. Army Medical Command... AND ACCOUNTS CLAIMS AGAINST THE UNITED STATES The Army Claims System § 536.12 Commanding General, U.S. Army Medical Command. (a) After consulting with the Commander USARCS on the selection of medical claims...

32 CFR 536.12 - Commanding General, U.S. Army Medical Command.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Commanding General, U.S. Army Medical Command... AND ACCOUNTS CLAIMS AGAINST THE UNITED STATES The Army Claims System § 536.12 Commanding General, U.S. Army Medical Command. (a) After consulting with the Commander USARCS on the selection of medical claims...

32 CFR 536.12 - Commanding General, U.S. Army Medical Command.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Commanding General, U.S. Army Medical Command... AND ACCOUNTS CLAIMS AGAINST THE UNITED STATES The Army Claims System § 536.12 Commanding General, U.S. Army Medical Command. (a) After consulting with the Commander USARCS on the selection of medical claims...

32 CFR 536.12 - Commanding General, U.S. Army Medical Command.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 3 2013-07-01 2013-07-01 false Commanding General, U.S. Army Medical Command... AND ACCOUNTS CLAIMS AGAINST THE UNITED STATES The Army Claims System § 536.12 Commanding General, U.S. Army Medical Command. (a) After consulting with the Commander USARCS on the selection of medical claims...

Rare intronic variants of TCF7L2 arising by selective sweeps in an indigenous population from Mexico.

PubMed

Acosta, Jose Luis; Hernández-Mondragón, Alma Cristal; Correa-Acosta, Laura Carolina; Cazañas-Padilla, Sandra Nathaly; Chávez-Florencio, Berenice; Ramírez-Vega, Elvia Yamilet; Monge-Cázares, Tulia; Aguilar-Salinas, Carlos A; Tusié-Luna, Teresa; Del Bosque-Plata, Laura

2016-05-26

Genetic variations of the TCF7L2 gene are associated with the development of Type 2 diabetes (T2D). The associated mutations have demonstrated an adaptive role in some human populations, but no studies have determined the impact of evolutionary forces on genetic diversity in indigenous populations from Mexico. Here, we sequenced and analyzed the variation of the TCF7L2 gene in three Amerindian populations and compared the results with whole-exon-sequencing of Mestizo populations from Sigma and the 1000 Genomes Project to assess the roles of selection and recombination in diversity. The diversity in the indigenous populations was biased to intronic regions. Most of the variation was low frequency. Only mutations rs77961654 and rs61724286 were located on exon 15. We did not observe variation in intronic region 4-6 in any of the three indigenous populations. In addition, we identified peaks of selective sweeps in the mestizo samples from the Sigma Project within this region. By replicating the analysis of association with T2D between case-controls from the Sigma Project, we determined that T2D was most highly associated with the rs7903146 risk allele and to a lesser extent with the other six variants. All associated markers were located in intronic region 4-6, and their r(2) values of linkage disequilibrium were significantly higher in the Mexican population than in Africans from the 1000 Genomes Project. We observed reticulations in both the haplotypes network analysis from seven marker associates and the neighborNet tree based on 6061 markers in the TCF7L2 gene identified from all samples of the 1000 Genomes Project. Finally, we identified two recombination hotspots in the upstream region and 3' end of the TCF7L2 gene. The lack of diversity in intronic region 4-6 in Indigenous populations could be an effect of selective sweeps generated by the selection of neighboring rare variants at T2D-associated mutations. The survivors' variants make the intronic region 4-6 the

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

Crystal structure of group II intron domain 1 reveals a template for RNA assembly

DOE PAGES

Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; ...

2015-10-26

Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. In this paper, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Composed of domain 1 from the Oceanobacillus iheyensis group II intron (266 nucleotides), this intermediate retains native-like features but adopts a compact conformation in which the active site cleft is closed. Transition between this closed andmore » the open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. Finally, the open state is then stabilized by sequential docking of downstream intron domains, suggesting a 'first come, first folded' strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures.« less

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.

cisprimertool: software to implement a comparative genomics strategy for the development of conserved intron scanning (CIS) markers.

PubMed

Jayashree, B; Jagadeesh, V T; Hoisington, D

2008-05-01

The availability of complete, annotated genomic sequence information in model organisms is a rich resource that can be extended to understudied orphan crops through comparative genomic approaches. We report here a software tool (cisprimertool) for the identification of conserved intron scanning regions using expressed sequence tag alignments to a completely sequenced model crop genome. The method used is based on earlier studies reporting the assessment of conserved intron scanning primers (called CISP) within relatively conserved exons located near exon-intron boundaries from onion, banana, sorghum and pearl millet alignments with rice. The tool is freely available to academic users at http://www.icrisat.org/gt-bt/CISPTool.htm. © 2007 ICRISAT.

The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

PubMed

Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

2015-05-01

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome. © 2015 Collins et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

PubMed Central

2013-01-01

Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC. Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them

Glycaemic control and hypoglycaemia with new insulin glargine 300 U/ml versus insulin glargine 100 U/ml in people with type 2 diabetes using basal insulin and oral antihyperglycaemic drugs: the EDITION 2 randomized 12-month trial including 6-month extension.

PubMed

Yki-Järvinen, H; Bergenstal, R M; Bolli, G B; Ziemen, M; Wardecki, M; Muehlen-Bartmer, I; Maroccia, M; Riddle, M C

2015-12-01

To compare the efficacy and safety of new insulin glargine 300 U/ml (Gla-300) with insulin glargine 100 U/ml (Gla-100) over 12 months of treatment in people with type 2 diabetes using basal insulin and oral antihyperglycaemic drugs (OADs). EDITION 2 (NCT01499095) was a randomized, 6-month, multicentre, open-label, two-arm, phase IIIa study investigating once-daily Gla-300 versus Gla-100, plus OADs (excluding sulphonylureas), with a 6-month safety extension. Similar numbers of participants in each group completed 12 months of treatment [Gla-300, 315 participants (78%); Gla-100, 314 participants (77%)]. The reduction in glycated haemoglobin was maintained for 12 months with both treatments: least squares (LS) mean (standard error) change from baseline -0.55 (0.06)% for Gla-300 and -0.50 (0.06)% for Gla-100; LS mean difference -0.06 [95% confidence interval (CI) -0.22 to 0.10)%]. A significant relative reduction of 37% in the annualized rate of nocturnal confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemia was observed with Gla-300 compared with Gla-100: rate ratio 0.63 [(95% CI 0.42-0.96); p = 0.031], and fewer participants experienced ≥1 event [relative risk 0.84 (95% CI 0.71-0.99)]. Severe hypoglycaemia was infrequent. Weight gain was significantly lower with Gla-300 than Gla-100 [LS mean difference -0.7 (95% CI -1.3 to -0.2) kg; p = 0.009]. Both treatments were well tolerated with a similar pattern of adverse events (incidence of 69 and 60% in the Gla-300 and Gla-100 groups). In people with type 2 diabetes treated with Gla-300 or Gla-100, and non-sulphonylurea OADs, glycaemic control was sustained over 12 months, with less nocturnal hypoglycaemia in the Gla-300 group. © 2015 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.

Discovering weighted patterns in intron sequences using self-adaptive harmony search and back-propagation algorithms.

PubMed

Huang, Yin-Fu; Wang, Chia-Ming; Liou, Sing-Wu

2013-01-01

A hybrid self-adaptive harmony search and back-propagation mining system was proposed to discover weighted patterns in human intron sequences. By testing the weights under a lazy nearest neighbor classifier, the numerical results revealed the significance of these weighted patterns. Comparing these weighted patterns with the popular intron consensus model, it is clear that the discovered weighted patterns make originally the ambiguous 5SS and 3SS header patterns more specific and concrete.

19 CFR 12.103 - Report to the U.S. Attorney.

Code of Federal Regulations, 2010 CFR

2010-04-01

... TREASURY SPECIAL CLASSES OF MERCHANDISE Switchblade Knives § 12.103 Report to the U.S. Attorney. Should... consideration of criminal prosecution. The port director shall hold the seized switchblade knives intact pending...

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout–derived cell line, RTG-2

USGS Publications Warehouse

Kurath, Gael; Purcell, Maureen K.; Wargo, Andrew; Park, Jeong Woo; Moon, Chang Hoon

2010-01-01

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout–derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

ExDom: an integrated database for comparative analysis of the exon–intron structures of protein domains in eukaryotes

PubMed Central

Bhasi, Ashwini; Philip, Philge; Manikandan, Vinu; Senapathy, Periannan

2009-01-01

We have developed ExDom, a unique database for the comparative analysis of the exon–intron structures of 96 680 protein domains from seven eukaryotic organisms (Homo sapiens, Mus musculus, Bos taurus, Rattus norvegicus, Danio rerio, Gallus gallus and Arabidopsis thaliana). ExDom provides integrated access to exon-domain data through a sophisticated web interface which has the following analytical capabilities: (i) intergenomic and intragenomic comparative analysis of exon–intron structure of domains; (ii) color-coded graphical display of the domain architecture of proteins correlated with their corresponding exon-intron structures; (iii) graphical analysis of multiple sequence alignments of amino acid and coding nucleotide sequences of homologous protein domains from seven organisms; (iv) comparative graphical display of exon distributions within the tertiary structures of protein domains; and (v) visualization of exon–intron structures of alternative transcripts of a gene correlated to variations in the domain architecture of corresponding protein isoforms. These novel analytical features are highly suited for detailed investigations on the exon–intron structure of domains and make ExDom a powerful tool for exploring several key questions concerning the function, origin and evolution of genes and proteins. ExDom database is freely accessible at: http://66.170.16.154/ExDom/. PMID:18984624

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.

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

Discovering Weighted Patterns in Intron Sequences Using Self-Adaptive Harmony Search and Back-Propagation Algorithms

PubMed Central

Wang, Chia-Ming; Liou, Sing-Wu

2013-01-01

A hybrid self-adaptive harmony search and back-propagation mining system was proposed to discover weighted patterns in human intron sequences. By testing the weights under a lazy nearest neighbor classifier, the numerical results revealed the significance of these weighted patterns. Comparing these weighted patterns with the popular intron consensus model, it is clear that the discovered weighted patterns make originally the ambiguous 5SS and 3SS header patterns more specific and concrete. PMID:23737711

Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays

PubMed Central

Sugnet, Charles W; Srinivasan, Karpagam; Clark, Tyson A; O'Brien, Georgeann; Cline, Melissa S; Wang, Hui; Williams, Alan; Kulp, David; Blume, John E; Haussler, David; Ares, Manuel

2006-01-01

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5′ splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. PMID:16424921

Crystallographic and magnetic properties of Cu2U-type hexaferrite

NASA Astrophysics Data System (ADS)

Kamishima, K.; Tajima, R.; Watanabe, K.; Kakizaki, K.; Fujimori, A.; Sakai, M.; Watanabe, K.; Abe, H.

2015-02-01

We have investigated the synthesis conditions, and the magnetic properties of the Cu2U-type hexagonal ferrite, Ba4Cu2Fe36O60. The Cu2U-type hexaferrite was synthesized at the sintering temperature of 1050 °C with the initial composition of Ba4.4Cu2Fe37.6O62.8 (Cu2U+0.2T-block). The saturation magnetizations at 300 K and 5 K are 46.8 A m2/kg and 65.0 A m2/kg, respectively. The Curie temperature is 420 °C which is lower than that of the M-type ferrite (450 °C) but higher than that of the Cu2Y-type ferrite (320 °C). The amount of the nonmagnetic impurity in this sample is estimated to be about 10 wt% by the electron probe micro analysis. We estimated the expected saturation magnetization to be 65.2 A m2/kg, by assuming the model of a Néel-type ferrimagnetic structure and the reduction of magnetization by the 10 wt% nonmagnetic impurity. This is consistent with the observed magnetization of 65.0 A m2/kg at 5 K.

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

Randomized Comparative Study of the U- and H-Type Approaches of the TVT-Secur Procedure for the Treatment of Female Stress Urinary Incontinence: One-Year Follow-Up

PubMed Central

Kim, Jung Jun; Lee, Young-Suk

2010-01-01

Purpose We compared outcomes of the U- and H-type approaches of the tension-free vaginal tape (TVT)-Secur procedure for the treatment of female stress urinary incontinence (SUI). Materials and Methods From March 2007 to July 2008, 115 women with SUI underwent TVT-Secur by a single surgeon. Patients were randomly assigned to either the U- or the H-type approach. After 12 months, postoperative changes in the Sandvik questionnaire, incontinence quality of life questionnaire (I-QoL), Bristol female lower urinary tract symptoms-scored form (BFLUTS-SF), and postoperative patient satisfaction were evaluated. Cure was regarded as no leakage on the Sandvik questionnaire. Complications were also evaluated. Results Of 115 women, 53 were treated with the U approach, and 62 women were treated with the H approach. At 12 months, 88.7% of those treated with the U approach and 87.1% of those treated with the H approach were cured (p=0.796). The I-QoL and filling, incontinence, sexual function, and QoL sum (BFLUTS-SF) scores were improved with both approaches, and there were no significant differences in the degree of improvement between approaches. Approximately 83.7% and 82.9% of the women treated with the U and H approaches, respectively, were satisfied with the outcome (p=0.858). There were 3 cases of intra-operative vaginal wall perforation in the H-type group. Immediate postoperative retention was observed in 2 women in the U-type group and 1 woman in the H-type group. One woman in the U-type group underwent tape releasing and cutting procedures for persistent large post-void residuals. Conclusions The U- and the H-type approaches of the TVT-Secur procedure provided comparable effectiveness for the treatment of female SUI. PMID:20428427

Molecular cloning of rat sperm galactosyl receptor, a C-type lectin with in vitro egg binding activity.

PubMed

Rivkin, E; Tres, L L; Kaplan-Kraicer, R; Shalgi, R; Kierszenbaum, A L

2000-07-01

Rat sperm galactosyl receptor is a member of the C-type animal lectin family showing preferential binding to N-acetylgalactosamine compared to galactose. Binding is mediated by a Ca(2+)-dependent carbohydrate-recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin receptor 2/3 (RHL-2/3). The molecular organization of the genomic DNA, cDNA, and derived amino acid sequence of rat testis galactosyl receptor have been determined and in vitro fertilization studies were conducted to ascertain its role. We have determined that the rat testis galactosyl receptor gene generates two mRNA species: one species, designated liver-type, is identical to RHL-2/3; the other, designated testis-type, contains one unspliced intron (86 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine-proline-aspartic acid/QPD) and flanked Ca(2+)-binding amino acid sequences were not present in the testis-type protein. Northern and Southern blots demonstrated presence of transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12-amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein species localized on the plasma membrane surface of the head and tail of rat sperm. Furthermore, capacitated rat sperm preincubated with polyclonal antisera to RHL-2/3 or to the CRD of the liver-type galactosyl receptor showed a statistically significant decrease in the in vitro fertilization rate. We conclude that rat sperm galactosyl receptor may play a role in egg binding and that an undetermined molecular mechanism operates to generate two proteins with identical intracellular amino terminal domain but only one of them displays a CRD and associated Ca(2+)-binding

Nucleotide sequence of the L1 ribosomal protein gene of Xenopus laevis: remarkable sequence homology among introns.

PubMed Central

Loreni, F; Ruberti, I; Bozzoni, I; Pierandrei-Amaldi, P; Amaldi, F

1985-01-01

Ribosomal protein L1 is encoded by two genes in Xenopus laevis. The comparison of two cDNA sequences shows that the two L1 gene copies (L1a and L1b) have diverged in many silent sites and very few substitution sites; moreover a small duplication occurred at the very end of the coding region of the L1b gene which thus codes for a product five amino acids longer than that coded by L1a. Quantitatively the divergence between the two L1 genes confirms that a whole genome duplication took place in Xenopus laevis approximately 30 million years ago. A genomic fragment containing one of the two L1 gene copies (L1a), with its nine introns and flanking regions, has been completely sequenced. The 5' end of this gene has been mapped within a 20-pyridimine stretch as already found for other vertebrate ribosomal protein genes. Four of the nine introns have a 60-nucleotide sequence with 80% homology; within this region some boxes, one of which is 16 nucleotides long, are 100% homologous among the four introns. This feature of L1a gene introns is interesting since we have previously shown that the activity of this gene is regulated at a post-transcriptional level and it involves the block of the normal splicing of some intron sequences. Images Fig. 3. Fig. 5. PMID:3841512

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

PubMed Central

Ajiro, Masahiko; Jia, Rong; Zhang, Lifang; Liu, Xuefeng; Zheng, Zhi-Ming

2012-01-01

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression. PMID:23056301

ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

PubMed

Jonsson, Frida; Westin, Ida Maria; Österman, Lennart; Sandgren, Ola; Burstedt, Marie; Holmberg, Monica; Golovleva, Irina

2018-02-20

Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable. © 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Glycaemic control and hypoglycaemia during 12 months of randomized treatment with insulin glargine 300 U/mL versus glargine 100 U/mL in people with type 1 diabetes (EDITION 4)

PubMed Central

Bergenstal, Richard M.; Bolli, Geremia B.; Ziemen, Monika; Rojeski, Maria; Espinasse, Melanie; Riddle, Matthew C.

2017-01-01

Aims Insulin glargine 300 U/mL (Gla‐300) offers a flatter pharmacodynamic profile than insulin glargine 100 U/mL (Gla‐100). We have compared these insulins over 1 year in people with type 1 diabetes (T1DM). Methods EDITION 4 was a 6‐month, multicentre, randomized, open‐label phase 3 study. People with T1DM who completed the 6 months continued randomized Gla‐300 or Gla‐100 once daily, morning or evening, for a further 6 months. Results Among 549 participants randomized, 444 completed the 12‐month study period (Gla‐300, 80%; Gla‐100, 82%). Mean HbA1c decreased similarly from baseline to month 12 in the 2 treatment groups (difference, 0.02 [95% CI, −0.13 to 0.17]) %‐units [0.2 (−1.5 to 1.9) mmol/mol]), to a mean of 7.86 %‐units (62.4 mmol/mol) in both groups. For morning vs evening injection, there was no difference in HbA1c change over 12 months for Gla‐100, but a significantly larger decrease in HbA1c was observed in the Gla‐300 morning group than in the Gla‐300 evening group (difference, −0.25 [−0.47 to −0.04] %‐units [−2.7 (−5.2 to −0.4) mmol/mol]). Mean glucose from the 8‐point SMPG profiles decreased from baseline, and was similar between the 2 treatment groups. Basal insulin dose was 20% higher with Gla‐300 than with Gla‐100, while hypoglycaemia event rates, analysed at night, over 24 hours, or according to different glycaemic thresholds, did not differ between treatment groups, regardless of injection time. Adverse event profiles did not differ between groups. Conclusions In T1DM, Gla‐300 provides glucose control comparable to that of Gla‐100, and can be given at any time of day. PMID:28661585

Glycaemic control and hypoglycaemia during 12 months of randomized treatment with insulin glargine 300 U/mL versus glargine 100 U/mL in people with type 1 diabetes (EDITION 4).

PubMed

Home, Philip D; Bergenstal, Richard M; Bolli, Geremia B; Ziemen, Monika; Rojeski, Maria; Espinasse, Melanie; Riddle, Matthew C

2018-01-01

Insulin glargine 300 U/mL (Gla-300) offers a flatter pharmacodynamic profile than insulin glargine 100 U/mL (Gla-100). We have compared these insulins over 1 year in people with type 1 diabetes (T1DM). EDITION 4 was a 6-month, multicentre, randomized, open-label phase 3 study. People with T1DM who completed the 6 months continued randomized Gla-300 or Gla-100 once daily, morning or evening, for a further 6 months. Among 549 participants randomized, 444 completed the 12-month study period (Gla-300, 80%; Gla-100, 82%). Mean HbA1c decreased similarly from baseline to month 12 in the 2 treatment groups (difference, 0.02 [95% CI, -0.13 to 0.17]) %-units [0.2 (-1.5 to 1.9) mmol/mol]), to a mean of 7.86 %-units (62.4 mmol/mol) in both groups. For morning vs evening injection, there was no difference in HbA1c change over 12 months for Gla-100, but a significantly larger decrease in HbA1c was observed in the Gla-300 morning group than in the Gla-300 evening group (difference, -0.25 [-0.47 to -0.04] %-units [-2.7 (-5.2 to -0.4) mmol/mol]). Mean glucose from the 8-point SMPG profiles decreased from baseline, and was similar between the 2 treatment groups. Basal insulin dose was 20% higher with Gla-300 than with Gla-100, while hypoglycaemia event rates, analysed at night, over 24 hours, or according to different glycaemic thresholds, did not differ between treatment groups, regardless of injection time. Adverse event profiles did not differ between groups. In T1DM, Gla-300 provides glucose control comparable to that of Gla-100, and can be given at any time of day. © 2017 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.

A numerical study of the supercritical CO2 plate heat exchanger subject to U-type, Z-type, and multi-pass arrangements

NASA Astrophysics Data System (ADS)

Zhu, Chen-Xi; Wang, Chi-Chuan

2018-01-01

This study proposes a numerical model for plate heat exchanger that is capable of handling supercritical CO2 fluid. The plate heat exchangers under investigation include Z-type (1-pass), U-type (1-pass), and 1-2 pass configurations. The plate spacing is 2.9 mm with a plate thickness of 0.8 mm, and the size of the plate is 600 mm wide and 218 mm in height with 60 degrees chevron angle. The proposed model takes into account the influence of gigantic change of CO2 properties. The simulation is first compared with some existing data for water-to-water plate heat exchangers with good agreements. The flow distribution, pressure drop, and heat transfer performance subject to the supercritical CO2 in plate heat exchangers are then investigated. It is found that the flow velocity increases consecutively from the entrance plate toward the last plate for the Z-type arrangement, and this is applicable for either water side or CO2 side. However, the flow distribution of the U-type arrangement in the water side shows opposite trend. Conversely, the flow distribution for U-type arrangement of CO2 depends on the specific flow ratio (C*). A lower C* like 0.1 may reverse the distribution, i.e. the flow velocity increases moderately alongside the plate channel like Z-type while a large C* of 1 would resemble the typical distribution in water channel. The flow distribution of CO2 side at the first and last plate shows a pronounced drop/surge phenomenon while the channels in water side does not reveal this kind of behavior. The performance of 2-pass plate heat exchanger, in terms of heat transfer rate, is better than that of 1-pass design only when C* is comparatively small (C* < 0.5). Multi-pass design is more effective when the dominant thermal resistance falls in the CO2 side.

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

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.

Microstructural characterization of annealed U-12Zr-4Pd and U-12Zr-4Pd-5Ln: Investigating Pd as a metallic fuel additive

NASA Astrophysics Data System (ADS)

Benson, Michael T.; He, Lingfeng; King, James A.; Mariani, Robert D.

2018-04-01

Palladium is being investigated as a potential additive to metallic fuel to control fuel-cladding chemical interaction (FCCI). A primary cause of FCCI is the lanthanide fission products moving to the fuel periphery and interacting with the cladding. This interaction will lead to wastage of the cladding and, given enough time or burn-up, eventually to a cladding breach. The current study is a scanning electron microscopy (SEM) and transmission electron microscopy (TEM) characterization of annealed U-12Zr-4Pd and U-12Zr-4Pd-5Ln, where Ln = 53Nd-25Ce-16Pr-6La. The present study shows that Pd preferentially binds the lanthanides over other fuel constituents, which may prevent lanthanide migration and interaction with the cladding during irradiation. The SEM analysis indicates the 1:1 Pd-Ln compound is being formed, while the TEM analysis, due to higher resolution, found the 1:1 compound, as well as Pd-rich compounds Pd2Ln and Pd3Ln2.

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

PubMed

Panda, Amaresh C; De, Supriyo; Grammatikakis, Ioannis; Munk, Rachel; Yang, Xiaoling; Piao, Yulan; Dudekula, Dawood B; Abdelmohsen, Kotb; Gorospe, Myriam

2017-07-07

High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs

PubMed Central

De, Supriyo; Grammatikakis, Ioannis; Munk, Rachel; Yang, Xiaoling; Piao, Yulan; Dudekula, Dawood B.; Gorospe, Myriam

2017-01-01

Abstract High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions. PMID:28444238

The utility of DNA sequences of an intron from the beta-fibrinogen gene in phylogenetic analysis of woodpeckers (Aves: Picidae).

PubMed

Prychitko, T M; Moore, W S

1997-10-01

Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies. Copyright 1997 Academic Press

Novel methodologies for spectral classification of exon and intron sequences

NASA Astrophysics Data System (ADS)

Kwan, Hon Keung; Kwan, Benjamin Y. M.; Kwan, Jennifer Y. Y.

2012-12-01

Digital processing of a nucleotide sequence requires it to be mapped to a numerical sequence in which the choice of nucleotide to numeric mapping affects how well its biological properties can be preserved and reflected from nucleotide domain to numerical domain. Digital spectral analysis of nucleotide sequences unfolds a period-3 power spectral value which is more prominent in an exon sequence as compared to that of an intron sequence. The success of a period-3 based exon and intron classification depends on the choice of a threshold value. The main purposes of this article are to introduce novel codes for 1-sequence numerical representations for spectral analysis and compare them to existing codes to determine appropriate representation, and to introduce novel thresholding methods for more accurate period-3 based exon and intron classification of an unknown sequence. The main findings of this study are summarized as follows: Among sixteen 1-sequence numerical representations, the K-Quaternary Code I offers an attractive performance. A windowed 1-sequence numerical representation (with window length of 9, 15, and 24 bases) offers a possible speed gain over non-windowed 4-sequence Voss representation which increases as sequence length increases. A winner threshold value (chosen from the best among two defined threshold values and one other threshold value) offers a top precision for classifying an unknown sequence of specified fixed lengths. An interpolated winner threshold value applicable to an unknown and arbitrary length sequence can be estimated from the winner threshold values of fixed length sequences with a comparable performance. In general, precision increases as sequence length increases. The study contributes an effective spectral analysis of nucleotide sequences to better reveal embedded properties, and has potential applications in improved genome annotation.

Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

PubMed

Li, Jun; Meng, Xiangbing; Zong, Yuan; Chen, Kunling; Zhang, Huawei; Liu, Jinxing; Li, Jiayang; Gao, Caixia

2016-09-12

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

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

Murata, Yasuhiko, E-mail: 97318@ib.k.u-tokyo.ac.jp; Furuyama, Isao; Oda, Shoji

2011-04-01

Highlights: {yields} A novel major transcript, AIFL-I4, is found. {yields} Nuclear localization of AIFL-I4 induces mitochondrial morphology change and suppression of cell proliferation. {yields} AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site does not induce these phenotypes. {yields} [2Fe-2S] cluster binding site is essential for these phenotypes. -- Abstract: Apoptosis-inducing factor-like (AIFL) protein contains a Rieske domain and pyridine nucleotide-disulfide oxidoreductase (Pyr{sub r}edox) domain that shows 35% homology to that of apoptosis-inducing factor (AIF) protein. We identified a novel major transcript of the medaka (Oryzias latipes) AIFL gene that retained intron 4 (AIFL-I4) in embryos and tissues frommore » adult fish. The product of this transcript, AIFL-I4 protein, lacked the Pyr{sub r}edox domain because of a nonsense codon in intron 4. Both AIFL-I4 and full-length AIFL (fAIFL) transcripts were highly expressed in the brain and late embryos, and relative fAIFL and AIFL-I4 expression levels differed among tissues. Transient expression of AIFL-I4 and fAIFL tagged with GFP showed that AIFL-I4 localized in the nucleus, while fAIFL localized throughout the cytoplasm. We also found that overexpression of AIFL-I4 induced a change in mitochondrial morphology and suppression of cell proliferation. AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site of the Rieske domain did not induce these phenotypes. This report is the first to demonstrate nuclear localization of a Rieske-type protein translated from the AIFL gene. Our data suggested that the [2Fe-2S] cluster binding site was essential for the nuclear localization and involved in mitochondrial morphology and suppression of cell proliferation.« less

A Novel Pathway for Sensory-Mediated Arousal Involves Splicing of an Intron in the period Clock Gene

PubMed Central

Cao, Weihuan; Edery, Isaac

2015-01-01

Study Objectives: D. melanogaster is an excellent animal model to study how the circadian (≅ 24-h) timing system and sleep regulate daily wake-sleep cycles. Splicing of a temperature-sensitive 3'-terminal intron (termed dmpi8) from the circadian clock gene period (per) regulates the distribution of daily activity in Drosophila. The role of dmpi8 splicing on daily behavior was further evaluated by analyzing sleep. Design: Transgenic flies of the same genetic background but expressing either a wild-type recombinant per gene or one where the efficiency of dmpi8 splicing was increased were exposed to different temperatures in daily light-dark cycles and sleep parameters measured. In addition, transgenic flies were briefly exposed to a variety of sensory-mediated stimuli to measure arousal responses. Results: Surprisingly, we show that the effect of dmpi8 splicing on daytime activity levels does not involve a circadian role for per but is linked to adjustments in sensory-dependent arousal and sleep behavior. Genetically altered flies with high dmpi8 splicing efficiency remain aroused longer following short treatments with light and non-photic cues such as mechanical stimulation. Conclusions: We propose that the thermal regulation of dmpi8 splicing acts as a temperature-calibrated rheostat in a novel arousal mechanism, so that on warm days the inefficient splicing of the dmpi8 intron triggers an increase in quiescence by decreasing sensory-mediated arousal, thus ensuring flies minimize being active during the hot midday sun despite the presence of light in the environment, which is usually a strong arousal cue for diurnal animals. Citation: Cao W, Edery I. A novel pathway for sensory-mediated arousal involves splicing of an intron in the period clock gene. SLEEP 2015;38(1):41–51. PMID:25325457

One-year sustained glycaemic control and less hypoglycaemia with new insulin glargine 300 U/ml compared with 100 U/ml in people with type 2 diabetes using basal plus meal-time insulin: the EDITION 1 12-month randomized trial, including 6-month extension.

PubMed

Riddle, M C; Yki-Järvinen, H; Bolli, G B; Ziemen, M; Muehlen-Bartmer, I; Cissokho, S; Home, P D

2015-09-01

To evaluate the maintenance of efficacy and safety of insulin glargine 300 U/ml (Gla-300) versus glargine 100 U/ml (Gla-100) in people with type 2 diabetes mellitus (T2DM) using basal plus meal-time insulin for 12 months in the EDITION 1 trial. EDITION 1 was a multicentre, randomized, open-label, two-arm, phase IIIa study. Participants completing the initial 6-month treatment period continued to receive Gla-300 or Gla-100, as previously randomized, once daily for a further 6-month open-label extension phase. Changes in glycated haemoglobin (HbA1c) and fasting plasma glucose concentrations, insulin dose, hypoglycaemic events and body weight were assessed. Of 807 participants enrolled in the initial phase, 89% (359/404) assigned to Gla-300 and 88% (355/403) assigned to Gla-100 completed 12 months. Glycaemic control was sustained in both groups (mean HbA1c: Gla-300, 7.24%; Gla-100, 7.42%), with more sustained HbA1c reduction for Gla-300 at 12 months: least squares mean difference Gla-300 vs Gla-100: HbA1c -0.17 [95% confidence interval (CI) -0.30 to -0.05]%. The mean daily basal insulin dose at 12 months was 1.03 U/kg for Gla-300 and 0.90 U/kg for Gla-100. Lower percentages of participants had ≥1 confirmed [≤3.9 mmol/l (≤70 mg/dl)] or severe hypoglycaemic event with Gla-300 than Gla-100 at any time of day [24 h; 86 vs 92%; relative risk 0.94 (95% CI 0.89-0.99)] and during the night [54 vs 65%; relative risk 0.84 (95% CI 0.75-0.94)], while the annualized rates of such hypoglycaemic events were similar. No between-treatment differences in adverse events were apparent. During 12 months of treatment of T2DM requiring basal and meal-time insulin, glycaemic control was better sustained and fewer individuals reported hypoglycaemia with Gla-300 than with Gla-100. The mean basal insulin dose was higher with Gla-300 compared with Gla-100, but total numbers of hypoglycaemic events and overall tolerability did not differ between treatments. © 2015 The Authors. Diabetes

A Historical Evaluation of the U12n Tunnel, Nevada National Security Site, Nye County, Nevada Part 1

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

Drollinger, Harold; Jones, Robert C; Bullard, Thomas F

2011-06-01

, ventilation equipment, air compressors, communications equipment, mining equipment, rail lines, retention ponds to impound tunnel effluent, and storage containers. Features on the mesa above the tunnel generally relate to tunnel ventilation and cooling, borehole drilling, and data recording facilities. Feature types include concrete foundations, instrument cable holes, drill holes, equipment pads, ventilation shafts, and ventilation equipment. The U12n Tunnel complex is eligible to the National Register of Historic Places under criteria a and c, consideration g of 36 CFR Part 60.4 as a historic landscape. Scientific research conducted at the tunnel has made significant contributions to the broad patterns of our history, particularly in regard to the Cold War era that was characterized by competing social, economic, and political ideologies between the former Soviet Union and the United States. The tunnel also possesses distinctive construction and engineering methods for conducting underground nuclear tests. The Desert Research Institute recommends that the U12n Tunnel area be left in place in its current condition and that the U12n Tunnel historic landscape be included in the NNSS monitoring program and monitored for disturbances or alterations on a regular basis.« less

Investigation of the estrogen receptor-alpha gene with type 2 diabetes and/or nephropathy in African-American and European-American populations.

PubMed

Gallagher, Carla J; Keene, Keith L; Mychaleckyj, Josyf C; Langefeld, Carl D; Hirschhorn, Joel N; Henderson, Brian E; Gordon, Candace J; Freedman, Barry I; Rich, Stephen S; Bowden, Donald W; Sale, Michèle M

2007-03-01

The estrogen receptor-alpha gene (ESR1) was selected as a positional candidate under a type 2 diabetes linkage peak at 6q24-27. A total of 42 ESR1 single nucleotide polymorphisms (SNPs) were genotyped in 380 African-American type 2 diabetic case subjects with end-stage renal disease (ESRD) and 276 African-American control subjects. A total of 22 ancestry informative markers were also genotyped, and the program Admixmap was used to adjust allelic and haplotypic association tests for individual estimates of admixture. The most significant association with type 2 diabetes-ESRD was with rs1033182 in intron 2 (P = 0.013, admixture-adjusted P(a) = 0.021). Genotyping 17 SNPs across a region of ESR1 intron 1-intron 2 in an expanded population of 851 case and 635 control subjects supported association with rs1033182 (P = 0.004, P(a) = 0.027) and with an independent six-SNP haplotype of high linkage disequilibrium spanning 6.4 kb (P < 0.0001, P(a) < 0.0001). The same 17 ESR1 SNPs were genotyped in 300 European-American type 2 diabetes-ESRD case subjects and 310 European-American control subjects. Two intron 2 SNPs, rs2431260 (P = 0.015) and rs1709183 (P = 0.019), and a four-SNP haplotype containing these SNPs (P = 0.033) were associated with type 2 diabetes and/or ESRD. Results suggest that intron 1 and intron 2 of the ESR1 gene may contain functionally important regions related to type 2 diabetes or ESRD risk.

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

SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

PubMed

Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

2015-04-01

Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Processing of Archaebacterial Intron-Containing tRNA Gene Transcripts

DTIC Science & Technology

1988-07-27

number) The overall goal of this project is to develop an understanding of tRNA gene structure and transcript processing in the halophilic Archaebacteria...containing precursor tRNAs in the halophilic Archaebecteria suggest that tRNATr p may be the only interrupted tR?4A gene in these organisms...1 August 1986 RESEARCH OBJECTIVE: To determine the mechanism of tRNA intron processing in the halophilic archaebacteria; characterize the enzyme

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

PubMed Central

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V.

2012-01-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3′-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. PMID:22661231

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.

PubMed

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V

2012-06-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

Computation of direct and inverse mutations with the SEGM web server (Stochastic Evolution of Genetic Motifs): an application to splice sites of human genome introns.

PubMed

Benard, Emmanuel; Michel, Christian J

2009-08-01

We present here the SEGM web server (Stochastic Evolution of Genetic Motifs) in order to study the evolution of genetic motifs both in the direct evolutionary sense (past-present) and in the inverse evolutionary sense (present-past). The genetic motifs studied can be nucleotides, dinucleotides and trinucleotides. As an example of an application of SEGM and to understand its functionalities, we give an analysis of inverse mutations of splice sites of human genome introns. SEGM is freely accessible at http://lsiit-bioinfo.u-strasbg.fr:8080/webMathematica/SEGM/SEGM.html directly or by the web site http://dpt-info.u-strasbg.fr/~michel/. To our knowledge, this SEGM web server is to date the only computational biology software in this evolutionary approach.

Structure of the human type IV collagen COL4A6 gene, which is mutated in Alport syndrome-associated leiomyomatosis

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

Zhang, Xu; Zhou, Jing; Reeders, S.T.

1996-05-01

Basement membrane (type IV) collagen, a subfamily of the collagen protein family, is encoded by six distinct genes in mammals. Three of those, COL4A3, COL4A4, and COL4A5, are linked with Alport syndrome (hereditary nephritis). Patients with leimoyomatosis associated with Alport syndrome have been shown to have deletions in the 5{prime} end of the COL4A6 gene, in addition to having deletions in COL4A6. The human COL4A6 gene is reported to be 425 kb as determined by mapping of overlapping YAC clones by probes for its 5{prime} and 3{prime} ends. In the present study we describe the complete exon/intron size pattern ofmore » the human COL4A6 gene. The 12 {lambda} phage clones characterized in the study spanned a total of 110 kb, including 85 kb of the actual gene and 25 kb of flanking sequences. The overlapping clones contained all 46 exons of the gene and all introns, except for intron 2. Since the total size of the exons and all introns except for intron 2 is about 85 kb, intron 2 must be about 340 kb. All exons of the gene were assigned to EcoRI restriction fragments to facilitate analysis of the gene in patients with leiomyomatosis associated with Alport syndrome. The exon size pattern of COL4A6 is highly homologous with that of the human and mouse COL4A2 genes, with 27 of the 46 exons of COL4A6 being identical in size between the genes. 42 refs., 2 figs., 3 tabs.« less

Nonsyndromic recessive deafness DFNB18 and Usher syndrome type IC are allelic mutations of USHIC.

PubMed

Ahmed, Zubair M; Smith, Tenesha N; Riazuddin, Saima; Makishima, Tomoko; Ghosh, Manju; Bokhari, Sirosh; Menon, Puthezhath S N; Deshmukh, Dilip; Griffith, Andrew J; Riazuddin, Sheikh; Friedman, Thomas B; Wilcox, Edward R

2002-06-01

Human chromosome 11 harbors two Usher type I loci, USHIB and USHIC, which encode myosin VIIA and harmonin, respectively. The USHIC locus overlaps the reported critical interval for nonsyndromic deafness locus DFNB18. We found an IVS12+5G-->C mutation in the USHIC gene, which is associated with nonsyndromic recessive deafness ( DFNB18) segregating in the original family, S-11/12. No other disease-associated mutation was found in the other 27 exons or in the intron-exon boundaries, and the IVS12+5G-->C mutation was not present in 200 representative unaffected individuals ascertained from the same area of India. An exon-trapping assay with a construct harboring IVS12+5G-->C generated wildtype spliced mRNA having exons 11 and 12 and mRNA that skipped exon 12. We conclude that mutations of USHIC can cause both Usher syndrome type IC and nonsyndromic recessive deafness DFNB18.

Glycaemic control and hypoglycaemia with insulin glargine 300 U/mL compared with glargine 100 U/mL in Japanese adults with type 2 diabetes using basal insulin plus oral anti-hyperglycaemic drugs (EDITION JP 2 randomised 12-month trial including 6-month extension).

PubMed

Terauchi, Y; Koyama, M; Cheng, X; Sumi, M; Riddle, M C; Bolli, G B; Hirose, T

2017-10-01

To compare insulin glargine 300 U/mL (Gla-300) with glargine 100 U/mL (Gla-100) in Japanese adults with uncontrolled type 2 diabetes on basal insulin and oral anti-hyperglycaemic drugs over 12 months. EDITION JP 2 was a randomised, open-label, phase 3 study. Following a 6-month treatment period, participants continued receiving previously assigned once daily Gla-300 or Gla-100, plus oral anti-hyperglycaemic drugs, in a 6-month extension period. Glycaemic control, hypoglycaemia and adverse events were assessed. The 12-month completion rate was 88% for Gla-300 and 96% for Gla-100, with comparable reasons for discontinuation. Mean HbA 1c decrease from baseline to month 12 was 0.3% in both groups. Annualised rates of confirmed (≤3.9mmol/L [≤70mg/dL]) or severe hypoglycaemia were lower with Gla-300 than Gla-100 (nocturnal [00:00-05:59h]: rate ratio 0.41; 95% confidence interval: 0.18 to 0.92; anytime [24h]: rate ratio 0.64; 95% confidence interval: 0.44 to 0.94). Cumulative number of hypoglycaemic events was lower with Gla-300 than Gla-100. Adverse event profiles were comparable between treatments. Over 12 months, Gla-300-treated participants achieved sustained glycaemic control and experienced less hypoglycaemia, particularly at night, versus Gla-100, supporting 6-month results. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival

PubMed Central

Fei, Dennis Liang; Motowski, Hayley; Chatrikhi, Rakesh; Gao, Shaojian; Kielkopf, Clara L.; Varmus, Harold

2016-01-01

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele. PMID:27776121

High yields of hydrogen production from methanol steam reforming with a cross-U type reactor

PubMed Central

Zhang, Shubin; Chen, Junyu; Zhang, Xuelin; Liu, Xiaowei

2017-01-01

This paper presents a numerical and experimental study on the performance of a methanol steam reformer integrated with a hydrogen/air combustion reactor for hydrogen production. A CFD-based 3D model with mass and momentum transport and temperature characteristics is established. The simulation results show that better performance is achieved in the cross-U type reactor compared to either a tubular reactor or a parallel-U type reactor because of more effective heat transfer characteristics. Furthermore, Cu-based micro reformers of both cross-U and parallel-U type reactors are designed, fabricated and tested for experimental validation. Under the same condition for reforming and combustion, the results demonstrate that higher methanol conversion is achievable in cross-U type reactor. However, it is also found in cross-U type reactor that methanol reforming selectivity is the lowest due to the decreased water gas shift reaction under high temperature, thereby carbon monoxide concentration is increased. Furthermore, the reformed gas generated from the reactors is fed into a high temperature proton exchange membrane fuel cell (PEMFC). In the test of discharging for 4 h, the fuel cell fed by cross-U type reactor exhibits the most stable performance. PMID:29121067

High yields of hydrogen production from methanol steam reforming with a cross-U type reactor.

PubMed

Zhang, Shubin; Zhang, Yufeng; Chen, Junyu; Zhang, Xuelin; Liu, Xiaowei

2017-01-01

This paper presents a numerical and experimental study on the performance of a methanol steam reformer integrated with a hydrogen/air combustion reactor for hydrogen production. A CFD-based 3D model with mass and momentum transport and temperature characteristics is established. The simulation results show that better performance is achieved in the cross-U type reactor compared to either a tubular reactor or a parallel-U type reactor because of more effective heat transfer characteristics. Furthermore, Cu-based micro reformers of both cross-U and parallel-U type reactors are designed, fabricated and tested for experimental validation. Under the same condition for reforming and combustion, the results demonstrate that higher methanol conversion is achievable in cross-U type reactor. However, it is also found in cross-U type reactor that methanol reforming selectivity is the lowest due to the decreased water gas shift reaction under high temperature, thereby carbon monoxide concentration is increased. Furthermore, the reformed gas generated from the reactors is fed into a high temperature proton exchange membrane fuel cell (PEMFC). In the test of discharging for 4 h, the fuel cell fed by cross-U type reactor exhibits the most stable performance.

Transformation from insulating p-type to semiconducting n-type conduction in CaCu3Ti4O12-related Na(Cu5/2Ti1/2)Ti4O12 ceramics

NASA Astrophysics Data System (ADS)

Li, Ming; Sinclair, Derek C.

2013-07-01

A double doping mechanism of Na+ + 1/2 Ti4+ → Ca2+ + 1/2 Cu2+ on the general formula Ca1-xNax(Cu3-x/2Tix/2)Ti4O12 has been used to prepare a series of isostructural CaCu3Ti4O12 (CCTO)-type perovskites. A complete solid solution exists for 0 ≤ x ≤ 1 and all compositions exhibit incipient ferroelectric behaviour with higher than expected intrinsic relative permittivity. Although CCTO ceramics typically exhibit n-type semiconductivity (room temperature, RT, resistivity of ˜10-100 Ω cm), Na(Cu5/2Ti1/2)Ti4O12 (NCTO) ceramics sintered at 950 °C consist of two insulating bulk phases (RT resistivity > 1 GΩ cm), one p-type and the other n-type. With increasing sintering temperature/period, the p-type phase transforms into the n-type phase. During the transformation, the resistivity and activation energy for electrical conduction (Ea ˜ 1.0 eV) of the p-type phase remain unchanged, whereas the n-type phase becomes increasingly conductive with Ea decreasing from ˜ 0.71 to 0.11 eV with increasing sintering temperature. These changes are attributed to small variations in stoichiometry that occur during high temperature ceramic processing with oxygen-loss playing a crucial role.

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.

Familial early-onset dementia with tau intron 10 + 16 mutation with clinical features similar to those of Alzheimer disease.

PubMed

Doran, Mark; du Plessis, Daniel G; Ghadiali, Eric J; Mann, David M A; Pickering-Brown, Stuart; Larner, Andrew J

2007-10-01

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) owing to the tau intron 10 + 16 mutation usually occurs with a prototypical frontotemporal dementia phenotype with prominent disinhibition and affective disturbances. To report a new FTDP-17 pedigree with the tau intron 10 + 16 mutation demonstrating a clinical phenotype suggestive of Alzheimer disease. Case reports. Regional neuroscience centers in northwest England. Patients We examined 4 members of a kindred in which 8 individuals were affected in 3 generations. All 4 patients reported memory difficulty. Marked anomia was also present, but behavioral disturbances were conspicuously absent in the early stages of disease. All patients had an initial clinical diagnosis of Alzheimer disease. No mutations were found in the presenilin or amyloid precursor protein genes. Pathologic examination of the proband showed features typical of FTDP-17, and tau gene analysis showed the intron 10 + 16 mutation. This pedigree illustrates the phenotypic variability of tau intron 10 + 16 mutations. In pedigrees with a clinical diagnosis of Alzheimer disease but without presenilin or amyloid precursor protein gene mutations, tau gene mutations may be found.

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

PubMed

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

2002-07-24

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

Paraoxonase promoter and intronic variants modify risk of sporadic amyotrophic lateral sclerosis

PubMed Central

Cronin, Simon; Greenway, Matthew J; Prehn, Jochen H M; Hardiman, Orla

2007-01-01

Background The paraoxonases, PON1–3, play a major protective role both against environmental toxins and as part of the antioxidant defence system. Recently, non‐synonymous coding single nucleotide polymorphisms (SNPs), known to lower serum PON activity, have been associated with sporadic ALS (SALS) in a Polish population. A separate trio based study described a detrimental allele at the PON3 intronic variant INS2+3651 (rs10487132). Association between PON gene cluster variants and SALS requires external validation in an independent dataset. Aims To examine the association of the promoter SNPs PON1−162G>A and PON1−108T>C; the non‐synonymous functional SNPs PON1Q192R and L55M and PON2C311S and A148G; and the intronic marker PON3INS2+3651A>G, with SALS in a genetically homogenous population. Methods 221 Irish patients with SALS and 202 unrelated control subjects were genotyped using KASPar chemistries. Statistical analyses and haplotype estimations were conducted using Haploview and Unphased software. Multiple permutation testing, as implemented in Unphased, was applied to haplotype p values to correct for multiple hypotheses. Results Two of the seven SNPs were associated with SALS in the Irish population: PON155M (OR 1.52, p = 0.006) and PON3INS2+3651 G (OR 1.36, p = 0.03). Two locus haplotype analysis showed association only when both of these risk alleles were present (OR 1.7, p = 0.005), suggesting a potential effect modification. Low functioning promoter variants were observed to influence this effect when compared with wild‐type. Conclusions These data provide additional evidence that genetic variation across the paroxanase loci may be common susceptibility factors for SALS. PMID:17702780

Rising incidence of type 2 diabetes in children in the U.K.

PubMed

Haines, Linda; Wan, Kay Chong; Lynn, Richard; Barrett, Timothy G; Shield, Julian P H

2007-05-01

To estimate the incidence of type 2 diabetes in children <17 years of age and to investigate the relationship of diabetes with increasing childhood obesity in the U.K. and the Republic of Ireland (ROI). Active monthly reporting of cases by consultant pediatricians occurred through the framework of the British Pediatric Surveillance Unit, with additional reports from specialist diabetes nurses. All children <17 years of age and diagnosed by their clinician as having non-type 1 diabetes from 1 October 2004 to 31 October 2005 were included. A total of 168 confirmed cases of non-type 1 diabetes were reported, resulting in a national incidence (excluding the ROI) of 1.3 x 100,000(-1) x year(-1). Of these, 40% were diagnosed with type 2 diabetes giving a minimum incidence of 0.53 x 100,000(-1) x year(-1). Children of ethnic minorities were greatly overrepresented, with those of black and South-Asian origin (England data only) having an incidence of 3.9 and 1.25 x 100,000(-1) x year(-1), respectively, compared with 0.35 x 100,000(-1) x year(-1) in those defined as white. Of those diagnosed with type 2 diabetes, 95% were overweight and 83% obese according to International Obesity Task Force guidelines. Eighty-four percent had a family history of type 2 diabetes. Type 2 diabetes is still less common than type 1 diabetes in U.K. children. However, compared with previous prevalence data, the frequency of type 2 diabetes appears to be increasing. Incidence among ethnic minorities is far higher than in whites, as previously described in the U.S. Increased adiposity and family history of type 2 diabetes were strongly associated with the diagnosis of type 2 diabetes in U.K. children.

Associations Between MAOA-uVNTR Genotype, Maltreatment, MAOA Methylation, and Alcohol Consumption in Young Adult Males.

PubMed

Bendre, Megha; Comasco, Erika; Checknita, Dave; Tiihonen, Jari; Hodgins, Sheilagh; Nilsson, Kent W

2018-03-01

Epigenetic mechanisms are candidate moderators of the effect of maltreatment on brain and behavior. Interactions between maltreatment and the monoamine oxidase A upstream variable number tandem repeat genotype (MAOA-uVNTR) are associated with alcohol-related problems. However, presently it is not known whether DNA methylation moderates this association. The study focused on 53 young adult males and aimed to determine whether MAOA methylation moderated the association of alcohol-related problems with the interaction of MAOA-uVNTR and maltreatment, and whether alcohol consumption moderated the association of MAOA methylation with the interaction of MAOA-uVNTR and maltreatment. MAOA-uVNTR genotypes with ≤ 3 and > 3 repeats were categorized as short (S) and long (L), respectively. Data on maltreatment were obtained retrospectively, using self-reported questionnaires. DNA methylation of 16 candidate CpGs within part of the MAOA first exon and intron was assessed and grouped based on principal component analyses. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test (AUDIT). Alcohol consumption was measured using AUDIT-C. Moderation effects were assessed and probed using the moderated moderation model and Johnson-Neyman's method, respectively. Carriers of the S allele, who experienced maltreatment and displayed lower Component 1 (mean of CpGs 13-16 in the first intron) MAOA methylation levels, reported higher AUDIT score in contrast to L-allele carriers. Carriers of the S allele, who reported higher AUDIT-C score and experienced maltreatment, displayed lower Component 3 (mean of CpGs 2-6 in the first exon) MAOA methylation levels than L-allele carriers. Intronic methylation moderated the association of alcohol-related problems with the interaction of MAOA-uVNTR and maltreatment. Alcohol consumption moderated the association of exonic methylation with the interaction of MAOA-uVNTR and maltreatment. These results suggest that

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes

PubMed Central

Wörheide, Gert

2017-01-01

Abstract One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. PMID:28633296

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.

78 FR 33790 - Endangered and Threatened Wildlife and Plants; 12-Month Findings on Petitions To Delist U.S...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-05

...-0077; 4500030115] Endangered and Threatened Wildlife and Plants; 12-Month Findings on Petitions To... Wildlife Service (``Service''), announce 12-month findings on two petitions to remove the U.S. captive-bred... review, we find that delisting the U.S. captive animals or U.S. captive-bred members of these species is...

[Vitamin B12 Deficiency in Type 2 Diabetes Mellitus].

PubMed

Tavares Bello, Carlos; Capitão, Ricardo Miguel; Sequeira Duarte, João; Azinheira, Jorge; Vasconcelos, Carlos

2017-10-31

Type 2 diabetes mellitus is a common disease, affecting up to 13.1% of the Portuguese population. In addition to the known micro and macrovascular complications, drug side effects constitute a major concern, leading to changes in the treatment guidelines, which favor safety over efficacy. Metformin is the first-line pharmacological treatment for most patients with type 2 diabetes mellitus; however, it has been associated with vitamin B12 deficiency in up to 30% of treated patients. The authors describe the prevalence of vitamin B12 deficiency in a diabetic population and explore the possible underlying factors. Retrospective, observational study. Clinical and laboratory data of type 2 diabetes mellitus patients whose vitamin B12 status was evaluated in the last decade (2005 - 2016) were analyzed. Patients with known malabsorptive syndromes or having undergone bariatric surgery were excluded from the study. Statistical analysis of the data was done and the results were considered statistically significant at p values < 0.05. The study included a total of 1007 patients (58% women) with a mean age of 66.4 ± 12.2 years and 11 ± 10.4 years of type 2 diabetes mellitus duration. These patients had a high prevalence of complications: diabetic renal disease 47.7%, neuropathy 9.2%, retinopathy 14.9%, coronary artery disease 8.4%, cerebrovascular disease 10.9%, and peripheral arterial disease 5.5%. Vitamin B12 deficiency (< 174 ng / dL) was present in 21.4% of the population and this subgroup was older (68.4 vs 65.8 years, p = 0.006), had a longer type 2 diabetes mellitus duration (13.35 vs 10.36 years; p = 0.001), higher prevalence of retinopathy (20.9% vs 13.3%; p = 0.005) and thyroid dysfunction (34% vs 23.7%; p = 0.002). Vitamin B12 deficiency was also more frequent in patients treated with metformin (24.7% vs 15.8%; p = 0.017), antiplatelet agents (25.4% vs 16.2%, p < 0.001), and calcium channel blockers (26.8% vs 18.2%; p = 0.001). After adjustment for possible

Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS.

PubMed

Luisier, Raphaelle; Tyzack, Giulia E; Hall, Claire E; Mitchell, Jamie S; Devine, Helen; Taha, Doaa M; Malik, Bilal; Meyer, Ione; Greensmith, Linda; Newcombe, Jia; Ule, Jernej; Luscombe, Nicholas M; Patani, Rickie

2018-05-22

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.

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.

Lusztig symmetries and Poincare-Birkhoff-Witt basis for wU{sub r,s}{sup d}(osp(1|2n))

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

Liu, Junli; College of Applied Sciences, Beijing University of Technology, Beijing 100124; Yang, Shilin

2013-12-15

We investigate a new kind of two-parameter weak quantized superalgebra wU{sub r,s}{sup d}(osp(1|2n)), which is a weak Hopf superalgebra. It has a homomorphic image which is isomorphic to the usual two-parameter quantum superalgebra U{sub r,s}(osp(1|2n)) of osp(1|2n). We also discuss the basis of wU{sub r,s}{sup d}(osp(1|2n)) by Lusztig's symmetries.

INSULIN GLARGINE 300 U/ML IS ASSOCIATED WITH LESS WEIGHT GAIN WHILE MAINTAINING GLYCEMIC CONTROL AND LOW RISK OF HYPOGLYCEMIA COMPARED WITH INSULIN GLARGINE 100 U/ML IN AN AGING POPULATION WITH TYPE 2 DIABETES.

PubMed

Munshi, Medha N; Gill, Jasvinder; Chao, Jason; Nikonova, Elena V; Patel, Meenakshi

2018-02-01

Assess efficacy, hypoglycemia, and weight gain in patients with type 2 diabetes (T2D) treated with insulin glargine 300 U/mL (Gla-300) or 100 U/mL (Gla-100) across different age groups. Pooled data were generated for patients randomized to Gla-300 or Gla-100 in the EDITION 2 (NCT01499095) and 3 (NCT01676220) studies. In 4 age groups (<55, ≥55 to <60, ≥60 to <65, ≥65 years), glycated hemoglobin A1C (A1C), percentage of patients reaching A1C <7.5% (58 mmol/mol), weight change, confirmed hypoglycemia (blood glucose ≤70 mg/dL), and/or severe hypoglycemia (events requiring third-party assistance) were analyzed with descriptive statistics and logistic, binomial, and analysis of covariance regression modeling. A1C reductions from baseline and proportions of patients at target were similar for Gla-300 and Gla-100 across all age groups at 6 and 12 months, but hypoglycemia incidence and event rate were lower with Gla-300 at 6 (both P<.001) and 12 months ( P<.001 and P = .005, respectively). Patients on Gla-300 gained less weight than those on Gla-100 at 6 ( P = .027) and 12 months ( P = .021). Changes in weight and daily weight-adjusted insulin dose decreased with increasing age at 6 ( P<.001 and P = .017, respectively) and 12 months ( P<.001 and P = .011, respectively). Older patients with T2D may benefit from treatment with Gla-300, which is associated with a lower hypoglycemia rate and less weight gain with similar efficacy compared with Gla-100. A1C = glycated hemoglobin A1C BMI = body mass index Gla-100 = insulin glargine 100 U/mL Gla-300 = insulin glargine 300 U/mL OAD = oral antidiabetes drug T2D = type 2 diabetes.

7 CFR 318.13-12 - Movement by the U.S. Department of Agriculture.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 5 2014-01-01 2014-01-01 false Movement by the U.S. Department of Agriculture. 318.13-12 Section 318.13-12 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE...

7 CFR 318.13-12 - Movement by the U.S. Department of Agriculture.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 5 2011-01-01 2011-01-01 false Movement by the U.S. Department of Agriculture. 318.13-12 Section 318.13-12 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE...

7 CFR 318.13-12 - Movement by the U.S. Department of Agriculture.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 5 2012-01-01 2012-01-01 false Movement by the U.S. Department of Agriculture. 318.13-12 Section 318.13-12 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE...

7 CFR 318.13-12 - Movement by the U.S. Department of Agriculture.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 5 2010-01-01 2010-01-01 false Movement by the U.S. Department of Agriculture. 318.13-12 Section 318.13-12 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE...

7 CFR 318.13-12 - Movement by the U.S. Department of Agriculture.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 5 2013-01-01 2013-01-01 false Movement by the U.S. Department of Agriculture. 318.13-12 Section 318.13-12 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE...

Amino acid substitutions and intron polymorphism of acetylcholinesterase1 associated with mevinphos resistance in diamondback moth, Plutella xylostella (L.).

PubMed

Yeh, Shih-Chia; Lin, Chia-Li; Chang, Cheng; Feng, Hai-Tung; Dai, Shu-Mei

2014-06-01

The diamondback moth, Plutella xylostella L., is the most destructive insect pest of Brassica crops in the world. It has developed resistance rapidly to almost every insecticide used for its control. Mevinphos, a fast degrading and slow resistance evocating organophosphorus insecticide, has been recommended for controlling P. xylostella in Taiwan for more than 40years. SHM strain of P. xylostella, with ca. 22-fold resistance to this chemical, has been established from a field SH strain by selecting with mevinphos since 1997. Three mutations, i.e., G892T, G971C, and T1156T/G leading to A298S, G324A, and F386F/V amino acid substitutions in acetylcholinesterase1 (AChE1), were identified in these two strains; along with three haplotype pairs and a polymorphic intron in AChE1 gene (ace1). Two genetically pure lines, i.e., an SHggt wild type with intron AS and an SHMTCN mutant carrying G892T, G971C, T1156T/G mutations and intron AR in ace1, were established by single pair mating and haplotype determination. The F1 of SHMTCN strain had 52-fold resistance to mevinphos in comparison with the F1 of SHggt strain. In addition, AChE1 of this SHMTCN population, which exhibited lower maximum velocity (Vmax) and affinity (Km), was less susceptible to the inhibition of mevinphos, with an I50 32-fold higher than that of the SHggt F1 population. These results imply that amino acid substitutions in AChE1 of SHMTCN strain are associated with mevinphos resistance in this insect pest, and this finding is important for insecticide resistance management of P. xylostella in the field. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

U.S. Crude Oil Production to 2025: Updated Projection of Crude Types

EIA Publications

2015-01-01

This report updates and extends a May 2014 EIA report, U.S. crude oil production forecast – analysis of crude types. It provides a projection of domestic crude oil production by crude type through 2025, supplementing the overall production projection provided in the AEO2015. Projections of production by crude type matter for several reasons. First, U.S. crude streams vary widely in quality. Second, the economics surrounding various options for the domestic use of additional domestic oil production are directly dependent on crude quality characteristics. Third, actual or potential export values also vary significantly with quality characteristics.

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

In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs

PubMed Central

Wu, Yong; Hu, Zhiqing; Li, Zhuo; Pang, Jialun; Feng, Mai; Hu, Xuyun; Wang, Xiaolin; Lin-Peng, Siyuan; Liu, Bo; Chen, Fangping; Wu, Lingqian; Liang, Desheng

2016-01-01

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23–26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition. PMID:26743572

In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.

PubMed

Wu, Yong; Hu, Zhiqing; Li, Zhuo; Pang, Jialun; Feng, Mai; Hu, Xuyun; Wang, Xiaolin; Lin-Peng, Siyuan; Liu, Bo; Chen, Fangping; Wu, Lingqian; Liang, Desheng

2016-01-08

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5' part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23-26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.

49 CFR 451.12 - Application for approval by design type.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 6 2013-10-01 2013-10-01 false Application for approval by design type. 451.12... Approval of New Containers § 451.12 Application for approval by design type. (a) For approval of new containers by design type, each application must include the following: (1) Engineering drawings and plans...

49 CFR 451.12 - Application for approval by design type.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 6 2014-10-01 2014-10-01 false Application for approval by design type. 451.12... Approval of New Containers § 451.12 Application for approval by design type. (a) For approval of new containers by design type, each application must include the following: (1) Engineering drawings and plans...

49 CFR 451.12 - Application for approval by design type.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 6 2012-10-01 2012-10-01 false Application for approval by design type. 451.12... Approval of New Containers § 451.12 Application for approval by design type. (a) For approval of new containers by design type, each application must include the following: (1) Engineering drawings and plans...

49 CFR 451.12 - Application for approval by design type.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 6 2010-10-01 2010-10-01 false Application for approval by design type. 451.12... Approval of New Containers § 451.12 Application for approval by design type. (a) For approval of new containers by design type, each application must include the following: (1) Engineering drawings and plans...

49 CFR 451.12 - Application for approval by design type.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 6 2011-10-01 2011-10-01 false Application for approval by design type. 451.12... Approval of New Containers § 451.12 Application for approval by design type. (a) For approval of new containers by design type, each application must include the following: (1) Engineering drawings and plans...

12. Photographic copy of photograph. (Source: U.S. Department of Interior. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. Photographic copy of photograph. (Source: U.S. Department of Interior. Office of Indian Affairs. Indian Irrigation Service. Annual Report, Fiscal Year 1925. Vol. I, Narrative and Photographs, Irrigation District #4, California and Southern Arizona, RG 75, Entry 655, Box 28, National Archives, Washington, DC.) Photographer unknown. SACATON DAM AND BRIDGE FROM QUARRY HILL, PRACTICALLY COMPLETED, 6/18/25 - San Carlos Irrigation Project, Sacaton Dam & Bridge, Gila River, T4S R6E S12/13, Coolidge, Pinal County, AZ

A Historical Evaluation of the U12n Tunnel, Nevada national Security Site, Nye County, Nevada Part 2 of 2

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

Drollinger, Harold; Jones, Robert C; Bullard, Thomas F

2011-06-01

, ventilation equipment, air compressors, communications equipment, mining equipment, rail lines, retention ponds to impound tunnel effluent, and storage containers. Features on the mesa above the tunnel generally relate to tunnel ventilation and cooling, borehole drilling, and data recording facilities. Feature types include concrete foundations, instrument cable holes, drill holes, equipment pads, ventilation shafts, and ventilation equipment. The U12n Tunnel complex is eligible to the National Register of Historic Places under criteria a and c, consideration g of 36 CFR Part 60.4 as a historic landscape. Scientific research conducted at the tunnel has made significant contributions to the broad patterns of our history, particularly in regard to the Cold War era that was characterized by competing social, economic, and political ideologies between the former Soviet Union and the United States. The tunnel also possesses distinctive construction and engineering methods for conducting underground nuclear tests. The Desert Research Institute recommends that the U12n Tunnel area be left in place in its current condition and that the U12n Tunnel historic landscape be included in the NNSS monitoring program and monitored for disturbances or alterations on a regular basis.« less

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

PubMed

Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

2003-08-14

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

12. General Arrangement Plan, Building 189D, U.S. Atomic Energy Commission, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. General Arrangement Plan, Building 189-D, U.S. Atomic Energy Commission, General Electric Company, Dwg. No. H-1-11068, 1958. - D-Reactor Complex, Deaeration Plant-Refrigeration Buildings, Area 100-D, Richland, Benton County, WA

EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies.

PubMed

Li, Chenhong; Riethoven, Jean-Jack M; Naylor, Gavin J P

2012-09-01

Recent innovations in next-generation sequencing have lowered the cost of genome projects. Nevertheless, sequencing entire genomes for all representatives in a study remains expensive and unnecessary for most studies in ecology, evolution and conservation. It is still more cost-effective and efficient to target and sequence single-copy nuclear gene markers for such studies. Many tools have been developed for identifying nuclear markers, but most of these have focused on particular taxonomic groups. We have built a searchable database, EvolMarkers, for developing single-copy coding sequence (CDS) and exon-primed-intron-crossing (EPIC) markers that is designed to work across a broad range of phylogenetic divergences. The database is made up of single-copy CDS derived from BLAST searches of a variety of metazoan genomes. Users can search the database for different types of markers (CDS or EPIC) that are common to different sets of input species with different divergence characteristics. EvolMarkers can be applied to any taxonomic group for which genome data are available for two or more species. We included 82 genomes in the first version of EvolMarkers and have found the methods to be effective across Placozoa, Cnidaria, Arthropod, Nematoda, Annelida, Mollusca, Echinodermata, Hemichordata, Chordata and plants. We demonstrate the effectiveness of searching for CDS markers within annelids and show how to find potentially useful intronic markers within the lizard Anolis. © 2012 Blackwell Publishing Ltd.

Incidence of type 1 and type 2 diabetes in youth in the U.S. Virgin Islands, 2001-2010.

PubMed

Washington, Raynard E; Orchard, Trevor J; Arena, Vincent C; Laporte, Ronald E; Tull, Eugene S

2013-06-01

To report the annual incidence of type 1 and type 2 diabetes among youth and to describe characteristics of youth diagnosed with diabetes in the U.S. Virgin Islands (USVI). All residents ≤19 years of age diagnosed with diabetes between January 2001 and December 2010 were identified from review of medical records of all hospitals and confirmed by physician query. A total of 82 eligible patients were identified and the registry ascertainment was estimated to be 98.7% complete. The overall age-adjusted annual incidence rates (per 100, 000) of type 1 and type 2 diabetes for the study period were 15.3 (95% CI: 11.3-20.1) and 9.6 (95% CI: 6.8-13.5), respectively. The incidence of type 1 diabetes increased significantly over the study period, with an epidemic-like threefold increase occurring from 2005 (8.7/100, 000) to 2006 (26.4/100, 000; p = 0.05). The incidence of type 1 diabetes was highest in the 10-19 age group in girls (25.6/100, 000), but no age difference was seen in boys, resulting from the lack of a pubertal peak in non-Hispanic Black boys. The incidence of type 2 diabetes rose significantly between 2001 (5.3/100, 000) and 2010 (12.5/100, 000; p = 0.03). The incidence of type 1 and type 2 diabetes in youth is increasing in the USVI, similar to global patterns. Further studies are needed to explore the missing pubertal rise in type 1 diabetes incidence in non-Hispanic Black boys and factors associated with the epidemic-like increases observed over the decade. © 2012 John Wiley & Sons A/S.

Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate.

PubMed

Geiss, K T; Abbas, G M; Makaroff, C A

1994-04-01

The mitochondrial gene coding for subunit 4 of the NADH dehydrogenase complex I (nad4) has been isolated and characterized from lettuce, Lactuca sativa. Analysis of nad4 genes in a number of plants by Southern hybridization had previously suggested that the intron content varied between species. Characterization of the lettuce gene confirms this observation. Lettuce nad4 contains two exons and one group IIA intron, whereas previously sequenced nad4 genes from turnip and wheat contain three group IIA introns. Northern analysis identified a transcript of 1600 nucleotides, which represents the mature nad4 mRNA and a primary transcript of 3200 nucleotides. Sequence analysis of lettuce and turnip nad4 cDNAs was used to confirm the intron/exon border sequences and to examine RNA editing patterns. Editing is observed at the 5' and 3' ends of the lettuce transcript, but is absent from sequences that correspond to exons two, three and the 5' end of exon four in turnip and wheat. In contrast, turnip transcripts are highly edited in this region, suggesting that homologous recombination of an edited and spliced cDNA intermediate was involved in the loss of introns two and three from an ancestral lettuce nad4 gene.

BIALLELIC POLYMORPHISM IN THE INTRON REGION OF B-TUBULIN GENE OF CRYPTOSPORIDIUM PARASITES

EPA Science Inventory

Nucleotide sequencing of polymerase chain reaction-amplified intron region of the Cryptosporidium parvum B-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is...

12. Architectural Floor Plans, 233S, U.S. Atomic Energy Commission, Hanford ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. Architectural Floor Plans, 233-S, U.S. Atomic Energy Commission, Hanford Atomic Products Operations, General Electric Company, Dwg. H-2-30464, 1956. - Reduction-Oxidation Complex, Plutonium Concentration Facility, 200 West Area, Richland, Benton County, WA

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

10 CFR 12.103 - Proceedings covered.

Code of Federal Regulations, 2011 CFR

2011-01-01

... under the Program Fraud Civil Remedies Act (31 U.S.C. 3801-12); (2) Any appeal of a decision made...” under 5 U.S.C. 504(b)(1)(C). (b) The Commission's failure to identify a type of proceeding as an...

Wild-type and mutated IDH1/2 enzymes and therapy responses.

PubMed

Molenaar, Remco J; Maciejewski, Jaroslaw P; Wilmink, Johanna W; van Noorden, Cornelis J F

2018-04-01

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are key enzymes in cellular metabolism, epigenetic regulation, redox states, and DNA repair. IDH1/2 mutations are causal in the development and/or progression of various types of cancer due to supraphysiological production of D-2-hydroxyglutarate. In various tumor types, IDH1/2-mutated cancers predict for improved responses to treatment with irradiation or chemotherapy. The present review discusses the molecular basis of the sensitivity of IDH1/2-mutated cancers with respect to the function of mutated IDH1/2 in cellular processes and their interactions with novel IDH1/2-mutant inhibitors. Finally, lessons learned from IDH1/2 mutations for future clinical applications in IDH1/2 wild-type cancers are discussed.

Differential intron retention in Jumonji chromatin modifier genes is implicated in reptile temperature-dependent sex determination

PubMed Central

Deveson, Ira W.; Holleley, Clare E.; Blackburn, James; Marshall Graves, Jennifer A.; Mattick, John S.; Waters, Paul D.; Georges, Arthur

2017-01-01

In many vertebrates, sex of offspring is determined by external environmental cues rather than by sex chromosomes. In reptiles, for instance, temperature-dependent sex determination (TSD) is common. Despite decades of work, the mechanism by which temperature is converted into a sex-determining signal remains mysterious. This is partly because it is difficult to distinguish the primary molecular events of TSD from the confounding downstream signatures of sexual differentiation. We use the Australian central bearded dragon, in which chromosomal sex determination is overridden at high temperatures to produce sex-reversed female offspring, as a unique model to identify TSD-specific features of the transcriptome. We show that an intron is retained in mature transcripts from each of two Jumonji family genes, JARID2 and JMJD3, in female dragons that have been sex-reversed by temperature but not in normal chromosomal females or males. JARID2 is a component of the master chromatin modifier Polycomb Repressive Complex 2, and the mammalian sex-determining factor SRY is directly regulated by an independent but closely related Jumonji family member. We propose that the perturbation of JARID2/JMJD3 function by intron retention alters the epigenetic landscape to override chromosomal sex-determining cues, triggering sex reversal at extreme temperatures. Sex reversal may then facilitate a transition from genetic sex determination to TSD, with JARID2/JMJD3 intron retention preserved as the decisive regulatory signal. Significantly, we also observe sex-associated differential retention of the equivalent introns in JARID2/JMJD3 transcripts expressed in embryonic gonads from TSD alligators and turtles, indicative of a reptile-wide mechanism controlling TSD. PMID:28630932

Differential intron retention in Jumonji chromatin modifier genes is implicated in reptile temperature-dependent sex determination.

PubMed

Deveson, Ira W; Holleley, Clare E; Blackburn, James; Marshall Graves, Jennifer A; Mattick, John S; Waters, Paul D; Georges, Arthur

2017-06-01

In many vertebrates, sex of offspring is determined by external environmental cues rather than by sex chromosomes. In reptiles, for instance, temperature-dependent sex determination (TSD) is common. Despite decades of work, the mechanism by which temperature is converted into a sex-determining signal remains mysterious. This is partly because it is difficult to distinguish the primary molecular events of TSD from the confounding downstream signatures of sexual differentiation. We use the Australian central bearded dragon, in which chromosomal sex determination is overridden at high temperatures to produce sex-reversed female offspring, as a unique model to identify TSD-specific features of the transcriptome. We show that an intron is retained in mature transcripts from each of two Jumonji family genes, JARID2 and JMJD3 , in female dragons that have been sex-reversed by temperature but not in normal chromosomal females or males. JARID2 is a component of the master chromatin modifier Polycomb Repressive Complex 2, and the mammalian sex-determining factor SRY is directly regulated by an independent but closely related Jumonji family member. We propose that the perturbation of JARID2/JMJD3 function by intron retention alters the epigenetic landscape to override chromosomal sex-determining cues, triggering sex reversal at extreme temperatures. Sex reversal may then facilitate a transition from genetic sex determination to TSD, with JARID2/JMJD3 intron retention preserved as the decisive regulatory signal. Significantly, we also observe sex-associated differential retention of the equivalent introns in JARID2/JMJD3 transcripts expressed in embryonic gonads from TSD alligators and turtles, indicative of a reptile-wide mechanism controlling TSD.

Ovarian Tumors related to Intronic Mutations in DICER1: A Report from the International Ovarian and Testicular Stromal Tumor Registry

PubMed Central

Schultz, Kris Ann; Harris, Anne; Messinger, Yoav; Sencer, Susan; Baldinger, Shari; Dehner, Louis P.; Hill, D. Ashley

2015-01-01

Germline DICER1 mutations have been described in individuals with pleuropulmonary blastoma (PPB), ovarian Sertoli-Leydig cell tumor (SLCT), sarcomas, multinodular goiter, thyroid carcinoma, cystic nephroma and other neoplastic conditions. Early results from the International Ovarian and Testicular Stromal Tumor Registry show germline DICER1 mutations in 48% of girls and women with SLCT. In this report, a young woman presented with ovarian undifferentiated sarcoma. Four years later, she presented with SLCT. She was successfully treated for both malignancies. Sequence results showed a germline intronic mutation in DICER1. This mutation results in an exact duplication of the six bases at the splice site at the intron 23 and exon 24 junction. Predicted improper splicing leads to inclusion of 10 bases of intronic sequence, frameshift and premature truncation of the protein disrupting the RNase IIIb domain. A second individual with SLCT was found to have an identical germline mutation. In each of the ovarian tumors, an additional somatic mutation in the RNase IIIb domain of DICER1 was found. In rare patients, germline intronic mutations in DICER1 that are predicted to cause incorrect splicing can also contribute to the pathogenesis of SLCT. PMID:26289771

The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize

PubMed Central

Bonen, Linda; Boer, Poppo H.; Gray, Michael W.

1984-01-01

We have determined the sequence of the wheat mitochondrial gene for cytochrome oxidase subunit II (COII) and find that its derived protein sequence differs from that of maize at only three amino acid positions. Unexpectedly, all three replacements are non-conservative ones. The wheat COII gene has a highly-conserved intron at the same position as in maize, but the wheat intron is 1.5 times longer because of an insert relative to its maize counterpart. Hybridization analysis of mitochondrial DNA from rye, pea, broad bean and cucumber indicates strong sequence conservation of COII coding sequences among all these higher plants. However, only rye and maize mitochondrial DNA show homology with wheat COII intron sequences and rye alone with intron-insert sequences. We find that a sequence identical to the region of the 5' exon corresponding to the transmembrane domain of the COII protein is present at a second genomic location in wheat mitochondria. These variations in COII gene structure and size, as well as the presence of repeated COII sequences, illustrate at the DNA sequence level, factors which contribute to higher plant mitochondrial DNA diversity and complexity. ImagesFig. 3.Fig. 4.Fig. 5. PMID:16453565

Identification of a deep intronic mutation in the COL6A2 gene by a novel custom oligonucleotide CGH array designed to explore allelic and genetic heterogeneity in collagen VI-related myopathies

PubMed Central

2010-01-01

Background Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. Methods We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. Results A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. Conclusions Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes. PMID:20302629

U.S. Environmental Protection Agency, Region 6 National Priorities List (NPL) Boundaries - 05/12/2014

EPA Pesticide Factsheets

Boundaries of sites in U.S. Environmental Protection Agency, Region 6 which are documented as being part of the National Priorities List as of May 12, 2014. The locations were determined by U.S. Environmental Protection Agency, Region 6 Superfund RPMs.

[U.S.-Mexico cross-border cooperation in research on diabetes mellitus type 2].

PubMed

Canela-Soler, Jaume; Frontini, María; Cerqueira, Maria Teresa; Ruiz-Holguín, Rosalba; Díaz-Apodaca, Beatriz A

2010-09-01

To describe and analyze, utilizing a case study approach, the U.S.- Mexico Border Diabetes Prevention and Control Project, a health research cooperation initiative incorporating the participation of federal, state, and local institutions of both countries. A model of equal representation, participation, consensus, and shared leadership was used, with the participation of more than 130 institutions. A sample of 4 020 people over 18 years of age was obtained by a random, multistage, stratified, clustered design. A questionnaire about diabetes mellitus type 2 (DM2) and health was applied. The statistical analysis took into account the design effect. The prevalence of diagnosed DM2 was 14.9% (95% confidence interval [95% CI]: 12.5-17.6) and the prevalence of diagnosed DM2 adjusted by age was 19.5% (95% CI: 16.8-22.6) on the Mexican side of the border and 16.1% (IC95%: 13.5-19.2) on the U.S. border side. There were differences between the DM2 prevalence and risk factors along the border. The U.S.-Mexico Border Diabetes Prevention and Control Project allowed the border zone between the two countries to be considered, for the first time ever, as a unit for epidemiological research. A shared understanding among all participating institutions and entities of sociopolitical structures and procedures is required for effective border health cooperation initiatives.

U.S.S. Hornet moves toward the Apollo 12 Command Module to retrieve it

NASA Image and Video Library

1969-11-24

U.S.S. Hornet, prime recovery vessel for the Apollo 12 lunar landing mission, moves toward the Apollo 12 Command Module to retrieve the spacecraft. A helicopter from the recovery ship, which took part in the recovery operations, hovers over the scene of the splashdown.

U.S. Environmental Protection Agency, Region 6 National Priorities List (NPL) Sites - 05/12/2014

EPA Pesticide Factsheets

Point locations for sites in U.S. Environmental Protection Agency, Region 6 which are documented as being part of the National Priorities List as of May 12, 2014. The locations were determined by U.S. Environmental Protection Agency, Region 6 Superfund RPMs.

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes.

PubMed

Francis, Warren R; Wörheide, Gert

2017-06-01

One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

44 CFR 78.12 - Eligible types of projects.

Code of Federal Regulations, 2012 CFR

2012-10-01

... OF HOMELAND SECURITY INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.12 Eligible types of projects. The following types of projects are eligible for.... (g) Minor physical flood mitigation projects that reduce localized flooding problems and do not...

10 CFR 12.103 - Proceedings covered.

Code of Federal Regulations, 2010 CFR

2010-01-01

... under the Program Fraud Civil Remedies Act (31 U.S.C. 3801-12); (2) Any appeal of a decision made... contract appeals as provided in section 8 of that Act (41 U.S.C. 607); and (3) Adversary adjudications...” under 5 U.S.C. 504(b)(1)(C). (b) The Commission's failure to identify a type of proceeding as an...

Phylogenetic inferences of Nepenthes species in Peninsular Malaysia revealed by chloroplast (trnL intron) and nuclear (ITS) DNA sequences.

PubMed

Bunawan, Hamidun; Yen, Choong Chee; Yaakop, Salmah; Noor, Normah Mohd

2017-01-26

The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus. Phylogeny reconstruction was carried out using neighbor-joining, maximum parsimony and Bayesian analyses. All the trees revealed two major clusters, a lowland group consisting of N. ampullaria, N. mirabilis, N. gracilis and N. rafflesiana, and another containing both intermediately distributed species (N. albomarginata and N. benstonei) and four highland species (N. sanguinea, N. macfarlanei, N. ramispina and N. alba). The trnL intron and ITS sequences proved to provide phylogenetic informative characters for deriving a phylogeny of Nepenthes species in Peninsular Malaysia. To our knowledge, this is the first molecular phylogenetic study of Nepenthes species occurring along an altitudinal gradient in Peninsular Malaysia.

[Frequency of intron 1 inversion of factor VIII gene in Chinese hemophilia A patients with case report of a female patient with heterozygous intron 1 inversion].

PubMed

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

2008-10-21

To investigate the frequency of intron 1 inversion (inv1) in FVIII gene in Chinese hemophilia A (HA) patients and to investigate the mechanism of pathogenesis. Peripheral blood samples were collected from 158 unrelated HA patients, aged 20 (1 - 73), including one female HA patient, aged 5, and several family members of a patient positive in inv1. One-stage method was used to assay the FVIII activity (FVIII:C). Long distance PCR and multiple PCR in duplex reactions were used to screen for the intron 22 inversion (inv22) and inv1 of the FVIII coding gene (F8). The F8 coding sequence was amplified with PCR and sequenced with an automatic sequencer. Two unrelated patients (pedigrees) were detected as inv1 positive with a positive rate of 1.26%. A rare female HA patient with inv1 was also discovered in a positive family (3 HA cases were found in this family and regarded as one case in calculating the total detection rate). The full length of FVIII was sequenced, and no other mutation was detected. There frequency of FVIII inv1 is low in Chinese HA patients compared with other populations. Female HA patients are heterozygous for FVIII inv1 and that may be resulted from nonrandom inactivation of X chromosome.

44 CFR 78.12 - Eligible types of projects.

Code of Federal Regulations, 2013 CFR

2013-10-01

..., DEPARTMENT OF HOMELAND SECURITY INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.12 Eligible types of projects. The following types of projects are eligible for.... (g) Minor physical flood mitigation projects that reduce localized flooding problems and do not...

44 CFR 78.12 - Eligible types of projects.

Code of Federal Regulations, 2014 CFR

2014-10-01

..., DEPARTMENT OF HOMELAND SECURITY INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.12 Eligible types of projects. The following types of projects are eligible for.... (g) Minor physical flood mitigation projects that reduce localized flooding problems and do not...

44 CFR 78.12 - Eligible types of projects.

Code of Federal Regulations, 2011 CFR

2011-10-01

..., DEPARTMENT OF HOMELAND SECURITY INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.12 Eligible types of projects. The following types of projects are eligible for.... (g) Minor physical flood mitigation projects that reduce localized flooding problems and do not...

44 CFR 78.12 - Eligible types of projects.

Code of Federal Regulations, 2010 CFR

2010-10-01

..., DEPARTMENT OF HOMELAND SECURITY INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.12 Eligible types of projects. The following types of projects are eligible for.... (g) Minor physical flood mitigation projects that reduce localized flooding problems and do not...

Nuclear sequestration of COL1A1 mRNA transcript associated with type I osteogenesis imperfecta (OI)

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

Primorac, D.; Stover, M.L.; McKinstry, M.B.

Previously we identified an OI type I patient with a splice donor mutation that resulted in intron 26 retention instead of exon skipping and sequestration of normal levels of the mutant transcript in the nuclear compartment. Intron retention was consistent with the exon definition hypothesis for splice site selection since the size of the exon-intron-exon unit was less than 300 bp. Furthermore, the retained intron contained in-frame stop codons which is thought to cause the mutant RNA to remain within the nucleus rather than appearing in the cytoplasm. To test these hypotheses, genomic fragments containing the normal sequence or themore » donor mutation were cloned into a collagen minigene and expressed in stably tansfected NIH 3T3 cells. None of the modifications to the normal intron altered the level of RNA that accumulated in the cytoplasm, as expected. However none of the modifications to the mutant intron allowed accumulation of normal levels of mRNA in the cytoplasm. Moreover, in contrast to our findings in the patient`s cells only low levels of mutant transcript were found in the nucleus; a fraction of the transcript did appear in the cytoplasm which had spliced the mutant donor site correctly. Nuclear run-on experiments demonstrated equal levels of transcription from each transgene. Expression of another donor mutation known to cause in-frame exon skipping in OI type IV was accurately reproduced in the minigene in transfected 3T3 cells. Our experience suggests that either mechanism can lead to formation of a null allele possibly related to the type of splicing events surrounding the potential stop codons. Understanding the rules governing inactivation of a collagen RNA transcript may be important in designing a strategy to inactivate a dominate negative mutation associated with the more severe forms of OI.« less

Quercetin Has Antimetastatic Effects on Gastric Cancer Cells via the Interruption of uPA/uPAR Function by Modulating NF-κb, PKC-δ, ERK1/2, and AMPKα.

PubMed

Li, Hai; Chen, Chen

2018-06-01

Gastric cancer (GC) is a malignancy with few effective treatment options after metastasis occurs. Quercetin (Qu) intake has been associated with reduced incidence and slow development of GC, probably due to its anti-proliferative and apoptotic effects, but it is unclear whether Qu can inhibit the metastatic activity. The urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system plays an important role in cancer metastasis. In this study, we measured both uPA activity and uPAR expression in GC and pericarcinous tissues, and we investigated the correlation between uPAR expression and the migratory and invasive activities of various GC cell lines. GC BGC823 and AGS cells were subjected to treatment with 10 μM Qu for 72 hours and uPAR knockdown, alone or in combination, before evaluating cell metastasis. The results showed that uPA activity and uPAR expression were higher in GC tissues than in pericarcinous tissues. Migratory and invasive activities of GC cell lines positively correlated with uPAR expression. Qu treatment decreased BGC823 and AGS cell migration and invasion, accompanied by reduced uPA and uPAR protein expression. Both Qu treatment and uPAR knockdown decreased matrix metalloproteinase-2 and -9 activity and blocked Pak1-Limk1-cofilin signaling. Qu treatment was associated with inhibition of NF-κb, PKC-δ, and ERK1/2, and with AMPKα activation. Specific inhibitors of NF-κb, PKC, and ERK1/2, and an AMPKα activator suppressed uPA and uPAR expression in GC cells. Collectively, Qu showed an antimetastatic effect on GC cells via the interruption of uPA/uPAR function and modulation of NF-κb, PKC-δ, ERK1/2, and AMPKα. This suggests that Qu is a promising agent against GC metastasis.

Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability.

PubMed

Joardar, Vinita; Abrams, Natalie F; Hostetler, Jessica; Paukstelis, Paul J; Pakala, Suchitra; Pakala, Suman B; Zafar, Nikhat; Abolude, Olukemi O; Payne, Gary; Andrianopoulos, Alex; Denning, David W; Nierman, William C

2012-12-12

The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25-36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population

12. Photocopy of photograph, U.S. Army, ca. 1943 (original print ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. Photocopy of photograph, U.S. Army, ca. 1943 (original print located at Rocky Mountain Arsenal, Commerce City, Colorado). R.M.A. - 321 - BOILER HOUSE 2nd FL. LOOKING EAST. - Rocky Mountain Arsenal, Boiler Plant-Central Gas Heat Plant, 1022 feet South of December Seventh Avenue; 525 feet West of D Street, Commerce City, Adams County, CO

12. Photocopy of photograph, U.S. Army, ca. 1943 (original print ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. Photocopy of photograph, U.S. Army, ca. 1943 (original print located at Rocky Mountain Arsenal, Commerce City, Colorado). R.M.A. - 742 - I.O.B. FILLING & STGE. LOOKING EAST. - Rocky Mountain Arsenal, Refrigeration Napalm & Incendiary Bomb Warehouse-Bomb Filling, 825 feet South of December Seventh Avenue; 2425 feet East of D Street, Commerce City, Adams County, CO

Understanding type Ia supernovae through their U-band spectra

NASA Astrophysics Data System (ADS)

Nordin, J.; Aldering, G.; Antilogus, P.; Aragon, C.; Bailey, S.; Baltay, C.; Barbary, K.; Bongard, S.; Boone, K.; Brinnel, V.; Buton, C.; Childress, M.; Chotard, N.; Copin, Y.; Dixon, S.; Fagrelius, P.; Feindt, U.; Fouchez, D.; Gangler, E.; Hayden, B.; Hillebrandt, W.; Kim, A.; Kowalski, M.; Kuesters, D.; Leget, P.-F.; Lombardo, S.; Lin, Q.; Pain, R.; Pecontal, E.; Pereira, R.; Perlmutter, S.; Rabinowitz, D.; Rigault, M.; Runge, K.; Rubin, D.; Saunders, C.; Smadja, G.; Sofiatti, C.; Suzuki, N.; Taubenberger, S.; Tao, C.; Thomas, R. C.; Nearby Supernova Factory

2018-06-01

Context. Observations of type Ia supernovae (SNe Ia) can be used to derive accurate cosmological distances through empirical standardization techniques. Despite this success neither the progenitors of SNe Ia nor the explosion process are fully understood. The U-band region has been less well observed for nearby SNe, due to technical challenges, but is the most readily accessible band for high-redshift SNe. Aims: Using spectrophotometry from the Nearby Supernova Factory, we study the origin and extent of U-band spectroscopic variations in SNe Ia and explore consequences for their standardization and the potential for providing new insights into the explosion process. Methods: We divide the U-band spectrum into four wavelength regions λ(uNi), λ(uTi), λ(uSi) and λ(uCa). Two of these span the Ca H&K λλ 3934, 3969 complex. We employ spectral synthesis using SYNAPPS to associate the two bluer regions with Ni/Co and Ti. Results: The flux of the uTi feature is an extremely sensitive temperature/luminosity indicator, standardizing the SN peak luminosity to 0.116 ± 0.011 mag root mean square (RMS). A traditional SALT2.4 fit on the same sample yields a 0.135 mag RMS. Standardization using uTi also reduces the difference in corrected magnitude between SNe originating from different host galaxy environments. Early U-band spectra can be used to probe the Ni+Co distribution in the ejecta, thus offering a rare window into the source of light curve power. The uCa flux further improves standardization, yielding a 0.086 ± 0.010 mag RMS without the need to include an additional intrinsic dispersion to reach χ2/dof 1. This reduction in RMS is partially driven by an improved standardization of Shallow Silicon and 91T-like SNe. All tables are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/614/A71. Individual SN spectra shown are available at http://snfactory.lbl.gov/snf/data

Leu72Met and Other Intronic Polymorphisms in the GHRL and GHSR Genes Are Not Associated with Type 2 Diabetes Mellitus, Insulin Resistance, or Serum Ghrelin Levels in a Saudi Population

PubMed Central

Joatar, Faris Elbahi; Al Qarni, Ali Ahmed; Ali, Muhalab E.; Al Masaud, Abdulaziz; Shire, Abdirashid M.; Das, Nagalla; Gumaa, Khalid

2017-01-01

Background Ghrelin (GHRL), a gastric peptide encoded by the GHRL gene, is known to be involved in energy homeostasis via its G protein receptor, encoded by the growth hormone secretagogue receptor (GHSR) gene. Some studies have shown associations between plasma GHRL levels and GHRL single-nucleotide polymorphisms (SNPs), namely the Leu72Met polymorphism (rs696217 TG), with type 2 diabetes mellitus (T2DM) and insulin resistance (IR), while others have not. The controversies in these associations raise the issue of ‘which SNPs in which populations.’ The aim of this study was to investigate whether SNPs in GHRL and/or GHSR genes were associated with T2DM, IR, or plasma GHRL levels among Arab Saudis. Methods Blood was collected from 208 Saudi subjects with (n=107) and without (n=101) T2DM. DNA samples from these subjects were analyzed by real-time polymerase chain reaction to genotype five intronic SNPs in the GHRL (rs696217 TG, rs27647 CT, rs2075356 CT, and rs4684677 AT) and GHSR (rs509030 GC) genes. In addition, plasma GHRL levels were measured by a radioimmunoassay. Results None of the SNPs were associated with T2DM, IR, or plasma GHRL levels. The frequencies of the alleles, genotypes, and haplotypes of the five SNPs were comparable between the T2DM patients and the non-diabetic subjects. A large number of the GHRL haplotypes indicates the molecular heterogeneity of the preproghrelin gene in this region. Conclusion Neither the Leu72Met polymorphism nor the other intronic GHRL and GHSR SNPs were associated with T2DM, IR, or GHRL levels. Further investigations should be carried out to explain the molecular basis of the association of the GHRL peptide with T2DM and IR. PMID:28956366

Leu72Met and Other Intronic Polymorphisms in the GHRL and GHSR Genes Are Not Associated with Type 2 Diabetes Mellitus, Insulin Resistance, or Serum Ghrelin Levels in a Saudi Population.

PubMed

Joatar, Faris Elbahi; Al Qarni, Ali Ahmed; Ali, Muhalab E; Al Masaud, Abdulaziz; Shire, Abdirashid M; Das, Nagalla; Gumaa, Khalid; Giha, Hayder A

2017-09-01

Ghrelin (GHRL), a gastric peptide encoded by the GHRL gene, is known to be involved in energy homeostasis via its G protein receptor, encoded by the growth hormone secretagogue receptor (GHSR) gene. Some studies have shown associations between plasma GHRL levels and GHRL single-nucleotide polymorphisms (SNPs), namely the Leu72Met polymorphism (rs696217 TG), with type 2 diabetes mellitus (T2DM) and insulin resistance (IR), while others have not. The controversies in these associations raise the issue of 'which SNPs in which populations.' The aim of this study was to investigate whether SNPs in GHRL and/or GHSR genes were associated with T2DM, IR, or plasma GHRL levels among Arab Saudis. Blood was collected from 208 Saudi subjects with (n=107) and without (n=101) T2DM. DNA samples from these subjects were analyzed by real-time polymerase chain reaction to genotype five intronic SNPs in the GHRL (rs696217 TG, rs27647 CT, rs2075356 CT, and rs4684677 AT) and GHSR (rs509030 GC) genes. In addition, plasma GHRL levels were measured by a radioimmunoassay. None of the SNPs were associated with T2DM, IR, or plasma GHRL levels. The frequencies of the alleles, genotypes, and haplotypes of the five SNPs were comparable between the T2DM patients and the non-diabetic subjects. A large number of the GHRL haplotypes indicates the molecular heterogeneity of the preproghrelin gene in this region. Neither the Leu72Met polymorphism nor the other intronic GHRL and GHSR SNPs were associated with T2DM, IR, or GHRL levels. Further investigations should be carried out to explain the molecular basis of the association of the GHRL peptide with T2DM and IR. Copyright © 2017 Korean Endocrine Society

20 CFR 361.12 - Procedures for salary offset: Types of collection.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Procedures for salary offset: Types of collection. 361.12 Section 361.12 Employees' Benefits RAILROAD RETIREMENT BOARD INTERNAL ADMINISTRATION... § 361.12 Procedures for salary offset: Types of collection. A debt will be collected in a lump sum or in...

The active gene that encodes human High Mobility Group 1 protein (HMG1) contains introns and maps to chromosome 13

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

Ferrari, S.; Finelli, P.; Rocchi, M.

The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 and q12, as determined by in situ hybridization. The mousemore » Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band. 18 refs., 3 figs.« less

Meal replacement reduces insulin requirement, HbA1c and weight long-term in type 2 diabetes patients with >100 U insulin per day.

PubMed

Kempf, K; Schloot, N C; Gärtner, B; Keil, R; Schadewaldt, P; Martin, S

2014-04-01

Despite high insulin doses, good glycaemic control is often lacking in type 2 diabetes patients and new therapeutic options are needed. In a proof of principle study, an energy-restricted, protein-rich meal replacement (PRMR) was examined as a means of reducing insulin requirement, HbA1C and body weight. Obese type 2 diabetes patients (n = 22) with >100 U insulin per day replaced, in week 1, the three main meals with 50 g of PRMR (Almased-Vitalkost) each (= 4903 kJ day(-1) ). In weeks 2-4, breakfast and dinner were replaced, and, in weeks 5-12, only dinner was replaced. Clinical parameters were determined at baseline, and after 4, 8 and 12 weeks, as well as after 1.5 years of follow-up. The Wilcoxon signed-rank test was used for the intention-to-treat analysis and the Mann-Whitney U-test for subgroup analyses. The 12-week-programme was completed by 15 participants (68%). After 1 week, the mean insulin dose was reduced from 147 (75) U to 91 (55) U day(-1) (P = 0.0001), and to 65 (32) U (P < 0.0001) after 12 weeks of study. Over a period of 12 weeks, HbA1c decreased from 8.8% (1.4%) to 8.1% (1.6%) (P = 0.048) and weight decreased from 118.0 (19.7) kg to 107.4 (19.2) kg (P < 0.0001). Moreover, body mass index, waist and hip circumference, fasting blood glucose, triglycerides and high-density lipoprotein cholesterol improved significantly. After 1.5 years, insulin requirement and weight remained significantly lower than baseline. Participants who continued PRMR further reduced their HbA1c, weight and insulin dose. Two patients were able to stop insulin therapy altogether. Energy-restricted PRMR was effective in reducing insulin requirement of type 2 diabetes patients with intensified insulin therapy accompanied by a reduction of HbA1c, weight and other cardiometabolic risk factors. With the continuous use of PRMR, glycaemic control might be improved in the long term. © 2013 The British Dietetic Association Ltd.

Conserved intron positions in FGFR genes reflect the modular structure of FGFR and reveal stepwise addition of domains to an already complex ancestral FGFR.

PubMed

Rebscher, Nicole; Deichmann, Christina; Sudhop, Stefanie; Fritzenwanker, Jens Holger; Green, Stephen; Hassel, Monika

2009-10-01

We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa.

Roles for herpes simplex virus type 1 U{sub L}34 and U{sub S}3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress

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

Bjerke, Susan L.; Roller, Richard J.

2006-04-10

Cells infected with wild type HSV-1 showed significant lamin A/C and lamin B rearrangement, while U{sub L}34-null virus-infected cells exhibited few changes in lamin localization, indicating that U{sub L}34 is necessary for lamin disruption. During HSV infection, U{sub S}3 limited the development of disruptions in the lamina, since cells infected with a U{sub S}3-null virus developed large perforations in the lamin layer. U{sub S}3 regulation of lamin disruption does not correlate with the induction of apoptosis. Expression of either U{sub L}34 or U{sub S}3 proteins alone disrupted lamin A/C and lamin B localization. Expression of U{sub L}34 and U{sub S}3more » together had little effect on lamin A/C localization, suggesting a regulatory interaction between the two proteins. The data presented in this paper argue for crucial roles for both U{sub L}34 and U{sub S}3 in regulating the state of the nuclear lamina during viral infection.« less

Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA

PubMed Central

Metz, Edward C.; Robles-Sikisaka, Refugio; Vacquier, Victor D.

1998-01-01

Strong positive Darwinian selection acts on two sperm fertilization proteins, lysin and 18-kDa protein, from abalone (Haliotis). To understand the phylogenetic context for this dramatic molecular evolution, we obtained sequences of mitochondrial cytochrome c oxidase subunit I (mtCOI), and genomic sequences of lysin, 18-kDa, and a G protein subunit. Based on mtDNA differentiation, four north Pacific abalone species diverged within the past 2 million years (Myr), and remaining north Pacific species diverged over a period of 4–20 Myr. Between-species nonsynonymous differences in lysin and 18-kDa exons exceed nucleotide differences in introns by 3.5- to 24-fold. Remarkably, in some comparisons nonsynonymous substitutions in lysin and 18-kDa genes exceed synonymous substitutions in mtCOI. Lysin and 18-kDa intron/exon segments were sequenced from multiple red abalone individuals collected over a 1,200-km range. Only two nucleotide changes and two sites of slippage variation were detected in a total of >29,000 nucleotides surveyed. However, polymorphism in mtCOI and a G protein intron was found in this species. This finding suggests that positive selection swept one lysin allele and one 18-kDa allele to fixation. Similarities between mtCOI and lysin gene trees indicate that rapid adaptive evolution of lysin has occurred consistently through the history of the group. Comparisons with mtCOI molecular clock calibrations suggest that nonsynonymous substitutions accumulate 2–50 times faster in lysin and 18-kDa genes than in rapidly evolving mammalian genes. PMID:9724763

TLR7 single-nucleotide polymorphisms in the 3' untranslated region and intron 2 independently contribute to systemic lupus erythematosus in Japanese women: a case-control association study

PubMed Central

2011-01-01

Introduction The Toll-like receptor 7 (TLR7) gene, encoded on human chromosome Xp22.3, is crucial for type I interferon production. A recent multicenter study in East Asian populations, comprising Chinese, Korean and Japanese participants, identified an association of a TLR7 single-nucleotide polymorphism (SNP) located in the 3' untranslated region (3' UTR), rs3853839, with systemic lupus erythematosus (SLE), especially in males, although some difference was observed among the tested populations. To test whether additional polymorphisms contribute to SLE in Japanese, we systematically analyzed the association of TLR7 with SLE in a Japanese female population. Methods A case-control association study was conducted on eight tag SNPs in the TLR7 region, including rs3853839, in 344 Japanese females with SLE and 274 healthy female controls. Results In addition to rs3853839, two SNPs in intron 2, rs179019 and rs179010, which were in moderate linkage disequilibrium with each other (r2 = 0.53), showed an association with SLE (rs179019: P = 0.016, odds ratio (OR) 2.02, 95% confidence interval (95% CI) 1.15 to 3.54; rs179010: P = 0.018, OR 1.75, 95% CI 1.10 to 2.80 (both under the recessive model)). Conditional logistic regression analysis revealed that the association of the intronic SNPs and the 3' UTR SNP remained significant after we adjusted them for each other. When only the patients and controls carrying the risk genotypes at the 3' UTR SNPpositionwere analyzed, the risk of SLE was significantly increased when the individuals also carried the risk genotypes at both of the intronic SNPs (P = 0.0043, OR 2.45, 95% CI 1.31 to 4.60). Furthermore, the haplotype containing the intronic risk alleles in addition to the 3' UTR risk allele was associated with SLE under the recessive model (P = 0.016, OR 2.37, 95% CI 1.17 to 4.80), but other haplotypes were not associated with SLE. Conclusions The TLR7 intronic SNPs rs179019 and rs179010 are associated with SLE independently of

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

PubMed

Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

2018-04-01

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

12 CFR 225.86 - What activities are permissible for any financial holding company?

Code of Federal Regulations, 2011 CFR

2011-01-01

... Board permits. (c) Activities permitted under section 4(k)(4) of the BHC Act (12 U.S.C. 1843(k)(4)). Any... Act (12 U.S.C. 1843(k)(4)(A) through (E), (H) and (I)). (d) Activities determined to be financial in... Bank Holding Company Act (12 U.S.C. 1843(k)(5)). (1) The following types of activities are financial in...

Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

PubMed

Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

2016-03-01

Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

A phylogeny of cycads (Cycadales) inferred from chloroplast matK gene, trnK intron, and nuclear rDNA ITS region.

PubMed

Chaw, Shu-Miaw; Walters, Terrence W; Chang, Chien-Chang; Hu, Shu-Hsuan; Chen, Shin-Hsiao

2005-10-01

Phylogenetic relationships among the three families and 12 living genera of cycads were reconstructed by distance and parsimony criteria using three markers: the chloroplast matK gene, the chloroplast trnK intron and the nuclear ITS/5.8S rDNA sequence. All datasets indicate that Cycadaceae (including only the genus Cycas) is remotely related to other cycads, in which Dioon was resolved as the basal-most clade, followed by Bowenia and a clade containing the remaining nine genera. Encephalartos and Lepidozamia are closer to each other than to Macrozamia. The African genus Stangeria is embedded within the New World subfamily Zamiodeae. Therefore, Bowenia is an unlikely sister to Stangeria, contrary to the view that they form the Stangeriaceae. The generic status of Dyerocycas and Chigua is unsupportable as they are paraphyletic with Cycas and the Zamia, respectively. Nonsense mutations in the matK gene and indels in the other two datasets lend evidence to reinforce the above conclusions. According to the phylogenies, the past geography of the genera of cycads and the evolution of character states are hypothesized and discussed. Within the suborder Zamiieae, Stangeria, and the tribe Zamieae evolved significantly faster than other genera. The matK gene and ITS/5.8S region contain more useful information than the trnK intron in addressing phylogeny. Redelimitations of Zamiaceae, Stangeriaceae, subfamily Encephalartoideae and subtribe Macrozamiineae are necessary.

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.

African Immigrants, the "New Model Minority": Examining the Reality in U.S. K-12 Schools

ERIC Educational Resources Information Center

Ukpokodu, Omiunota N.

2018-01-01

African immigrants in the U.S. have been headlined as America's "new model minority." The purpose of this paper is to examine if evidence exists to support the claim of African immigrant students' (AIS) educational achievement and excellence (a core indicator of the "model minority" theory) in U.S. k-12 schools. Using a…

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.

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

Friction stir processing on high carbon steel U12

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

Tarasov, S. Yu., E-mail: tsy@ispms.ru; Rubtsov, V. E., E-mail: rvy@ispms.ru; National Research Tomsk Polytechnic University, Tomsk, 634050

2015-10-27

Friction stir processing (FSP) of high carbon steel (U12) samples has been carried out using a milling machine and tools made of cemented tungsten carbide. The FSP tool has been made in the shape of 5×5×1.5 mm. The microstructural characterization of obtained stir zone and heat affected zone has been carried out. Microhardness at the level of 700 MPa has been obtained in the stir zone with microstructure consisting of large grains and cementitte network. This high-level of microhardness is explained by bainitic reaction developing from decarburization of austenitic grains during cementite network formation.

Suprafamilial relationships among Rodentia and the phylogenetic effect of removing fast-evolving nucleotides in mitochondrial, exon and intron fragments.

PubMed

Montgelard, Claudine; Forty, Ellen; Arnal, Véronique; Matthee, Conrad A

2008-11-26

The number of rodent clades identified above the family level is contentious, and to date, no consensus has been reached on the basal evolutionary relationships among all rodent families. Rodent suprafamilial phylogenetic relationships are investigated in the present study using approximately 7600 nucleotide characters derived from two mitochondrial genes (Cytochrome b and 12S rRNA), two nuclear exons (IRBP and vWF) and four nuclear introns (MGF, PRKC, SPTBN, THY). Because increasing the number of nucleotides does not necessarily increase phylogenetic signal (especially if the data is saturated), we assess the potential impact of saturation for each dataset by removing the fastest-evolving positions that have been recognized as sources of inconsistencies in phylogenetics. Taxonomic sampling included multiple representatives of all five rodent suborders described. Fast-evolving positions for each dataset were identified individually using a discrete gamma rate category and sites belonging to the most rapidly evolving eighth gamma category were removed. Phylogenetic tree reconstructions were performed on individual and combined datasets using Parsimony, Bayesian, and partitioned Maximum Likelihood criteria. Removal of fast-evolving positions enhanced the phylogenetic signal to noise ratio but the improvement in resolution was not consistent across different data types. The results suggested that elimination of fastest sites only improved the support for nodes moderately affected by homoplasy (the deepest nodes for introns and more recent nodes for exons and mitochondrial genes). The present study based on eight DNA fragments supports a fully resolved higher level rodent phylogeny with moderate to significant nodal support. Two inter-suprafamilial associations emerged. The first comprised a monophyletic assemblage containing the Anomaluromorpha (Anomaluridae + Pedetidae) + Myomorpha (Muridae + Dipodidae) as sister clade to the Castorimorpha (Castoridae + Geomyoidea

Suprafamilial relationships among Rodentia and the phylogenetic effect of removing fast-evolving nucleotides in mitochondrial, exon and intron fragments

PubMed Central

2008-01-01

Background The number of rodent clades identified above the family level is contentious, and to date, no consensus has been reached on the basal evolutionary relationships among all rodent families. Rodent suprafamilial phylogenetic relationships are investigated in the present study using ~7600 nucleotide characters derived from two mitochondrial genes (Cytochrome b and 12S rRNA), two nuclear exons (IRBP and vWF) and four nuclear introns (MGF, PRKC, SPTBN, THY). Because increasing the number of nucleotides does not necessarily increase phylogenetic signal (especially if the data is saturated), we assess the potential impact of saturation for each dataset by removing the fastest-evolving positions that have been recognized as sources of inconsistencies in phylogenetics. Results Taxonomic sampling included multiple representatives of all five rodent suborders described. Fast-evolving positions for each dataset were identified individually using a discrete gamma rate category and sites belonging to the most rapidly evolving eighth gamma category were removed. Phylogenetic tree reconstructions were performed on individual and combined datasets using Parsimony, Bayesian, and partitioned Maximum Likelihood criteria. Removal of fast-evolving positions enhanced the phylogenetic signal to noise ratio but the improvement in resolution was not consistent across different data types. The results suggested that elimination of fastest sites only improved the support for nodes moderately affected by homoplasy (the deepest nodes for introns and more recent nodes for exons and mitochondrial genes). Conclusion The present study based on eight DNA fragments supports a fully resolved higher level rodent phylogeny with moderate to significant nodal support. Two inter-suprafamilial associations emerged. The first comprised a monophyletic assemblage containing the Anomaluromorpha (Anomaluridae + Pedetidae) + Myomorpha (Muridae + Dipodidae) as sister clade to the Castorimorpha

Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

PubMed Central

Calvanese, Vincenzo; Mallya, Meera; Campbell, R Duncan; Aguado, Begoña

2008-01-01

Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F) undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C) and not on their own. PMID:18817541

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

The State of Wiki Usage in U.S. K-12 Schools: Leveraging Web 2.0 Data Warehouses to Assess Quality and Equity in Online Learning Environments

ERIC Educational Resources Information Center

Reich, Justin; Murnane, Richard; Willett, John

2012-01-01

To document wiki usage in U.S. K-12 settings, this study examined a representative sample drawn from a population of nearly 180,000 wikis. The authors measured the opportunities wikis provide for students to develop 21st-century skills such as expert thinking, complex communication, and new media literacy. The authors found four types of wiki…

12. Photographic copy of photograph. (Source: U.S. Department of Interior. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. Photographic copy of photograph. (Source: U.S. Department of Interior. Office of Indian Affairs. Indian Irrigation Service. Annual Report, Fiscal Year 1925. Vol. I, Narrative and Photographs, Irrigation District #4, California and Southern Arizona, RG 75, Entry 655, Box 28, National Archives, Washington, DC.) Photographer unknown. PIMA LATERAL, LINING EQUIPMENT, 5/13/25 - San Carlos Irrigation Project, Pima Lateral, Main Canal at Sacaton Dam, Coolidge, Pinal County, AZ