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Sample records for 5prime splice donor

  1. 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

    SciTech Connect

    Willing, M.; Deschenes, S.

    1994-09-01

    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 exon 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.

  2. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

    SciTech Connect

    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi )

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  3. Insertion of part of an intron into the 5[prime] untranslated region of a Caenorhabditis elegans gene converts it into a trans-spliced gene

    SciTech Connect

    Conrad, R.; Thomas, J.; Spieth, J.; Blumenthal, T. )

    1991-04-01

    In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis elegans, there are two SLs, Sl1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. The authors demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5[prime] splice site into the 5[prime] untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of a vit-5 intron, including the 3[prime] splice site, were inserted into the 5[prime] untranslated region of a vit-2/vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-2/vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. They termed the region of a trans-spliced mRNA precursor between the 5[prime] end and the first 3[prime] splice site an 'outrun'. The results suggest that if a transcript begins with intronlike sequence followed by a 3[prime] splice site, this alone may constitute an outrun and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.

  4. Alternatively spliced exons encode the tissue-specific 5{prime} termini of leukocyte pp52 and stromal cell S37 mRNA isoforms

    SciTech Connect

    Thompson, A.A.; May, W.; Denny, C.T.

    1996-03-05

    The pp52 gene encodes an intracellular, F-actin-binding phosphoprotein (also designated LSP1 and WP34) postulated to function in cytoskeleton dynamics and cell motility. We previously reported that different mRNA isoforms are expressed from this gene in cells of the leukocyte lineage versus mesodermally derived cells. These tissue-specific mRNA isoforms are identical except for 5{prime}-untranslated regions and sequences coding for unique N-termini of 23 and 21 amino acids, respectively. As this is a single-copy gene, we predicted that these tissue-specific mRNA isoforms would be generated by alternative RNA splicing. We report that the unique 5{prime} sequences in these mRNA isoforms are encoded in two separate exons containing ATG initiation codes. These features confirm that the pp52 and S37 mRNA isoforms are generated by alternative RNA splicing and establish that they are independently translated. Other results presented here indicate that the differential expression of these exons in leukocytes versus mesodermally derived cells is regulated at the level of transcription by tissue-specific promoters. 30 refs., 2 figs., 1 tab.

  5. Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals

    PubMed Central

    2010-01-01

    Background Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals. Results Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts. Conclusions Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts. PMID:21062477

  6. Thermodynamic Modeling of Donor Splice Site Recognition in pre-mRNA

    NASA Astrophysics Data System (ADS)

    Aalberts, Daniel P.; Garland, Jeffrey A.

    2004-03-01

    When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 snRNA with the donor (5') splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our Finding with Binding method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

  7. Thermodynamic modeling of donor splice site recognition in pre-mRNA

    NASA Astrophysics Data System (ADS)

    Garland, Jeffrey A.; Aalberts, Daniel P.

    2004-04-01

    When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 small nuclear RNA with the donor ( 5' ) splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our “finding with binding” method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

  8. Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

    SciTech Connect

    Solera, J. ); Magallon, M.; Martin-Villar, J. ); Coloma, A. )

    1992-02-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

  9. A computational approach for prediction of donor splice sites with improved accuracy.

    PubMed

    Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Rao, A R; Wahi, S D

    2016-09-01

    Identification of splice sites is important due to their key role in predicting the exon-intron structure of protein coding genes. Though several approaches have been developed for the prediction of splice sites, further improvement in the prediction accuracy will help predict gene structure more accurately. This paper presents a computational approach for prediction of donor splice sites with higher accuracy. In this approach, true and false splice sites were first encoded into numeric vectors and then used as input in artificial neural network (ANN), support vector machine (SVM) and random forest (RF) for prediction. ANN and SVM were found to perform equally and better than RF, while tested on HS3D and NN269 datasets. Further, the performance of ANN, SVM and RF were analyzed by using an independent test set of 50 genes and found that the prediction accuracy of ANN was higher than that of SVM and RF. All the predictors achieved higher accuracy while compared with the existing methods like NNsplice, MEM, MDD, WMM, MM1, FSPLICE, GeneID and ASSP, using the independent test set. We have also developed an online prediction server (PreDOSS) available at http://cabgrid.res.in:8080/predoss, for prediction of donor splice sites using the proposed approach. PMID:27302911

  10. A novel donor splice-site mutation of major intrinsic protein gene associated with congenital cataract in a Chinese family

    PubMed Central

    Zeng, Lu; Liu, Wenqiang; Feng, Wenguo; Wang, Xing; Dang, Hui; Gao, Luna; Yao, Jing

    2013-01-01

    Purpose To identify the disease-causing gene in a Chinese family with autosomal dominant congenital cataract. Methods Clinical and ophthalmologic examinations were performed on all members of a Chinese family with congenital cataract. Nine genes associated with congenital cataract were screened using direct DNA sequencing. Mutations were confirmed using restriction fragment length polymorphism (RFLP) analysis. The mutated major intrinsic protein (MIP) minigene, which carries the disease-causing splice-site mutation, and the wild-type (WT) MIP minigene were constructed using the pcDNA3.1 expression vector. Wild-type and mutant MIP minigene constructs were transiently transfected into HeLa cells. After 48 h of incubation at 37 °C, total RNA isolation and reverse transcription (RT)–PCR analysis were performed, and PCR products were separated and confirmed with sequencing. Results Direct DNA sequence analysis identified a novel splice-site mutation in intron 3 (c.606+1 G>A) of the MIP gene. To investigate the manner in which the splice donor mutation could affect mRNA splicing, WT and mutant MIP minigenes were inserted in the pcDNA3.1 (+) vector. Constructs were transfected into HeLa cells. RT–PCR analysis showed that the donor splice site mutation led to deletion of exon 3 in the mRNA encoded by the MIP gene. Conclusions The present study identified a novel donor splice-site mutation (c.606+1G>A) in the MIP gene in a Chinese family with congenital cataract. In vitro RT–PCR analysis showed that this splice-site mutation resulted in the deletion of exon 3 from mRNA encoded by the MIP gene. This is the first report to show that donor splice-site mutation in MIP gene can cause autosomal dominant congenital cataract. PMID:24319327

  11. A novel splice donor site mutation in EPHA2 caused congenital cataract in a Chinese family

    PubMed Central

    Bu, Juan; He, Sijie; Wang, Lejin; Li, Jiankang; Liu, Jing; Zhang, Xiuqing

    2016-01-01

    Background: Congenital cataract is a rare disorder characterized by crystallin denaturation, which becomes a major cause of childhood blindness. Although more than fifty pathogenic genes for congenital cataract have been reported, the genetic causes of many cataract patients remain unknown. In this study, the aim is to identify the genetic cause of a five-generation Chinese autosomal dominant congenital cataract family. Methods: Whole exome sequencing (WES) was performed on three affected and one unaffected member of the family, known causative genes were scanned first. Sanger sequencing was used to validate co-segregation of the candidate variant in the family. The impact on the transcript and amino acid sequences of the variant was further analyzed. Results: We identified a novel splice donor site mutation c. 2825+1G >A in EPHA2 that was absent in public and in-house databases and showed co-segregation in the family. This variant resulted in an altered splice that led to protein truncation. Conclusions: The mutation we identified was responsible for congenital cataract in our studied family. Our findings broaden the spectrum of causative mutations in EPHA2 gene for congenital cataract and suggest that WES is an efficient strategy to scan variants in known causative genes for genetically heterogeneous diseases. PMID:27380975

  12. A novel splice donor site at nt 1534 is required for long-term maintenance of HPV31 genomes

    SciTech Connect

    Poppelreuther, Sven; Iftner, Thomas; Stubenrauch, Frank

    2008-01-05

    Human papillomaviruses (HPV) are small double-stranded DNA viruses that replicate as low copy number nuclear plasmids during the persistent phase. HPV only possess nine open reading frames but extend their coding capabilities by alternative RNA splicing. We have identified in cell lines with replicating HPV31 genomes viral transcripts that connect the novel splice donor (SD) sites at nt 1426 and 1534 within the E1 replication gene to known splice acceptors at nt 3295 or 3332 within the E2/E4 region. These transcripts are polyadenylated and are present at low amounts in the non-productive and productive phase of the viral life cycle. Mutation of the novel splice sites in the context of HPV31 genomes revealed that the inactivation of SD1534 had only minor effects in short-term replication assays but displayed a low copy number phenotype in long-term cultures which might be due to the expression of alternative E1 circumflex E4 or yet unknown viral proteins. This suggests a regulatory role for minor splice sites within E1 for papillomavirus replication.

  13. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal

    PubMed Central

    Mueller, Nancy; Das, Atze T.; Berkhout, Ben

    2016-01-01

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5′ splice site (5′ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5′ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5′ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations. PMID:27455303

  14. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal.

    PubMed

    Mueller, Nancy; Das, Atze T; Berkhout, Ben

    2016-01-01

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations. PMID:27455303

  15. Utilisation of a cryptic non-canonical donor splice site of the gene encoding PARAFIBROMIN is associated with familial isolated primary hyperparathyroidism

    PubMed Central

    Bradley, K; Cavaco, B; Bowl, M; Harding, B; Young, A; Thakker, R

    2005-01-01

    More than 99% of all splice sites conform to consensus sequences that usually include the invariant dinucleotides gt and ag at the 5' and 3' ends of the introns, respectively. We report on the utilisation of a non-consensus (non-canonical) donor splice site within exon 1 of the HRPT2 gene in familial isolated primary hyperparathyroidism (FIHP). HRPT2 mutations are more frequently associated with the hyperparathyroidism-jaw tumour syndrome (HPT-JT). Patients with FIHP were identified to have a donor splice site mutation, IVS1+1 g→a, and the consequences of this for RNA processing were investigated. The mutant mRNA lacked 30 bp and DNA sequence analysis revealed this to result from utilisation of an alternative cryptic non-canonical donor splice site (gaatgt) in exon 1 together with the normally occurring acceptor splice site in intron 1. Translation of this mutant mRNA predicted the in-frame loss of 10 amino acids in the encoded protein, termed PARAFIBROMIN. Thus, these FIHP patients are utilising a ga-ag splice site pair, which until recently was considered to be incompatible with splicing but is now known to occur as a rare (<0.02%) normal splicing variant. PMID:16061557

  16. Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

    PubMed

    Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

    2015-12-15

    We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis. PMID:26671123

  17. The cyclization of arabinosyladenine-5-prime-phosphorimidazolide

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1991-01-01

    When arabinosyladenine-5-prime-phosphorimidazolide is allowed to decompose in aqueous solution at room temperature and pH 7.2, depending on the buffer, 5-24 percent is converted to the 2-prime,5-prime-cyclic phosphate (V). Although the extent of cyclization is much greater than for adenosine-5-prime-phosphorimidazolide, cyclization is less efficient than hydrolysis and so would not substantially decrease the efficiency of condensation reactions in aqueous solution. The significance of this result for prebiotic chemistry is discussed.

  18. Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

    SciTech Connect

    Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard; Bies, Juraj; Wolff, Linda; Mougel, Marylene . E-mail: mmougel@univ-montp1.fr

    2004-12-20

    Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.

  19. Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag.

    PubMed

    Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard; Bies, Juraj; Wolff, Linda; Mougel, Marylène

    2004-12-20

    Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism. PMID:15567434

  20. Isolation of an insulin-like growth factor II cDNA with a unique 5 prime untranslated region from human placenta

    SciTech Connect

    Shen, Shujane; Daimon, Makoto; Wang, Chunyeh; Ilan, J. ); Jansen, M. )

    1988-03-01

    Human insulin-like growth factor II (IGF-II) cDNA from a placental library was isolated and sequenced. The 5{prime} untranslated region (5{prime}-UTR) sequence of this cDNA differs completely from that of adult human liver and has considerable base sequence identity to the same region of an IGF-II cDNA of a rat liver cell line, BRL-3A. Human placental poly(A){sup +} RNA was probed with either the 5{prime}-UTR of the isolated human placental IGF-II cDNA or the 5{prime}-UTR of the IGF-II cDNA obtained from adult human liver. No transcripts were detected by using the 5{prime}-UTR of the adult liver IGF-II as the probe. In contrast, three transcripts of 6.0, 3.2, and 2.2 kilobases were detected by using the 5{prime}-UTR of the placental IGF-II cDNA as the probe or the probe from the coding sequence. A fourth IGF-II transcript of 4.9 kilobases presumably containing a 5{prime}-UTR consisting of a base sequence dissimilar to that of either IGF-II 5{prime}-UTR was apparent. Therefore, IGF-II transcripts detected may be products of alternative splicing as their 5{prime}-UTR sequence is contained within the human IGF-II gene or they may be a consequence of alternative promoter utilization in placenta.

  1. Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

    PubMed Central

    Solera, J; Magallón, M; Martin-Villar, J; Coloma, A

    1992-01-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment. Images Figure 1 PMID:1346483

  2. Escape variants of the XPR1 gammaretrovirus receptor are rare due to reliance on a splice donor site and a short hypervariable loop

    PubMed Central

    Lu, Xiaoyu; Martin, Carrie; Bouchard, Christelle; Kozak, Christine A.

    2014-01-01

    Entry determinants in the XPR1 receptor for the xenotropic/polytropic mouse leukemia viruses (XP-MLVs) lie in its third and fourth putative extracellular loops (ECLs). The critical ECL3 receptor determinant overlies a splice donor and is evolutionarily conserved in vertebrate XPR1 genes; 2 of the 3 rare replacement mutations at this site destroy this receptor determinant. The 13 residue ECL4 is hypervariable, and replacement mutations carrying an intact ECL3 site alter but do not abolish receptor activity, including replacement of the entire loop with that of a jellyfish (Cnidaria) XPR1. Because ECL4 deletions are found in all X-MLV-infected Mus subspecies, we deleted each ECL4 residue to determine if deletion-associated restriction is residue-specific or is effected by loop size. All deletions influence receptor function, although different deletions affect different XP-MLVs. Thus, receptor usage of a constrained splice site and a loop that tolerates mutations severely limits the likelihood of host escape mutations. PMID:25151060

  3. A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR

    PubMed Central

    Broen, Kelly; Levenga, Henriette; Vos, Johanna; van Bergen, Kees; Fredrix, Hanny; Greupink-Draaisma, Annelies; Kester, Michel; Falkenburg, J. H. Frederik; de Witte, Theo; Griffioen, Marieke; Dolstra, Harry

    2011-01-01

    Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT. PMID:21738768

  4. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

    PubMed Central

    Hagiwara, Y.; Nishio, H.; Kitoh, Y.; Takeshima, Y.; Narita, N.; Wada, H.; Yokoyama, M.; Nakamura, H.; Matsuo, M.

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. Images Figure 2 Figure 5 PMID:8279470

  5. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

    PubMed

    Hagiwara, Y; Nishio, H; Kitoh, Y; Takeshima, Y; Narita, N; Wada, H; Yokoyama, M; Nakamura, H; Matsuo, M

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. PMID:8279470

  6. Splicing defects in the COL3A1 gene: marked preference for 5' (donor) spice-site mutations in patients with exon-skipping mutations and Ehlers-Danlos syndrome type IV.

    PubMed Central

    Schwarze, U; Goldstein, J A; Byers, P H

    1997-01-01

    Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis. Images Figure 1 Figure 3 Figure 6 PMID:9399899

  7. Oligomerizations of deoxyadenosine bis-phosphates and of their 3-prime-5-prime, 3-prime-3-prime, and 5-prime-5-prime dimers - Effects of a pyrophosphate-linked, poly(T) analog

    NASA Technical Reports Server (NTRS)

    Visscher, J.; Bakker, C. G.; Schwartz, Alan W.

    1990-01-01

    The effect of a 3-prime-5-prime pyrophosphate-linked oligomer of pTp on oligomerizations of pdAp and of its 3-prime-5-prime, 3-prime-3-prime, and 5-prime-5-prime dimers was investigated, using HPLC to separate the reaction mixtures; peak detection was by absorbance monitoring at 254 nm. It was expected that the dimers would form stable complexes with the template, with the degree of stability depending upon the internal linkage of each dimer. It was found that, although the isomers differ substantially in their oligomerization behavior in the absence of template, the analog-template catalyzes the oligomerization to about the same extent in all three cases.

  8. A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signaling pathway and causes Lenz microphthalmia syndrome

    PubMed Central

    Esmailpour, Taraneh; Riazifar, Hamidreza; Liu, Linan; Donkervoort, Sandra; Huang, Vincent H; Madaan, Shreshtha; Shoucri, Bassem M; Busch, Anke; Wu, Jie; Towbin, Alexander; Chadwick, Robert B; Sequeira, Adolfo; Vawter, Marquis P; Sun, Guoli; Johnston, Jennifer J; Biesecker, Leslie G; Kawaguchi, Riki; Sun, Hui; Kimonis, Virginia; Huang, Taosheng

    2014-01-01

    Introduction Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition. Methods and results Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells. Conclusions We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway. PMID:24431331

  9. Soluble minerals in chemical evolution. II - Characterization of the adsorption of 5-prime-AMP and 5-prime-CMP on a variety of soluble mineral salts

    NASA Technical Reports Server (NTRS)

    Chan, Stephen; Orenberg, James; Lahav, Noam

    1987-01-01

    The adsorption of 5-prime-AMP and 5-prime-CMP is studied in the saturated solutions of several mineral salts as a function of pH, ionic strength, and surface area of the solid salt. It is suggested that the adsorption which results from the binding between the nucleotide molecule and the salt surface is due to electrostatic forces. The adsorption is reversible in nature and decreases with increasing ionic strength.

  10. HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

    SciTech Connect

    Tan, Chye Ling; Gunaratne, Jayantha; Lai, Deborah; Carthagena, Laetitia; Wang, Qian; Xue, Yue Zhen; Quek, Ling Shih; Doorbar, John; Bachelerie, Francoise; Thierry, Francoise; Bellanger, Sophie

    2012-07-20

    The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

  11. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  12. 5[prime] to 3[prime] nucleic acid synthesis using 3[prime]-photoremovable protecting group

    DOEpatents

    Pirrung, M.C.; Shuey, S.W.; Bradley, J.C.

    1999-06-01

    The present invention relates, in general, to a method of synthesizing a nucleic acid, and, in particular, to a method of effecting 5[prime] to 3[prime] nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5[prime] end. The invention also relates to a method of effecting mutation analysis using such arrays. The invention further relates to compounds and compositions suitable for use in such methods.

  13. Effect of cold exposure on thyroxine 5 prime -deiodinase activity in iron-deficient rats

    SciTech Connect

    Brigham, D.E.; Beard, J.L. )

    1991-03-11

    When exposed to cold, severely anemic iron-deficient (ID) rats become hypothermic, fail to adequately increase metabolic rate, and have lower interscapular brown adipose tissue (IBAT) thyroxine 5{prime}-deiodinase (5{prime}-DI) activity compared to control (CN) rats. Less severely anemic rats and CN rats were exposed to 4C for 6h. Rectal temperatures were measured hourly, and VO{sub 2} was measured both prior to and throughout the cold exposure period. IBAT 5{prime}-DI activity was measured in cold-exposed ID and CN rats and compared to ID and CN rats that were not cold exposed. During cold exposure, both ID and CN rats increased metabolic rate similarly. IBAT 5{prime}-DI activity also increased similarly in both groups after cold exposure. These results show that moderately anemic DI rats acutely increase metabolic rate and IBAT 5{prime}-DI activity in the cold. This suggests brown fat production of thyroid hormone is not limiting thermoregulatory performance.

  14. Template-directed oligomerization of 5-prime-deoxy 5-nucleosideacetic acid derivatives

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1990-01-01

    5-prime-deoxy 5-nucleosideacetic acids H-II are isostructural analogs of nucleotides with a carboxylate group in the place of the 5-prime-phosphate group. In this work, their oligomerization in aqueous solution was studied using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogs IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.

  15. Splicing fidelity

    PubMed Central

    Koodathingal, Prakash; Staley, Jonathan P.

    2013-01-01

    The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5′ splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5′ splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5′ splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5′ splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell. PMID:23770752

  16. Interleukin-5 Priming of Human Eosinophils Alters Siglec-8–Mediated Apoptosis Pathways

    PubMed Central

    Nutku-Bilir, Esra; Hudson, Sherry A.; Bochner, Bruce S.

    2008-01-01

    Previously, we have identified the sequential activation of reactive oxygen species (ROS), mitochondria, and caspase-3, -8, and -9, in Siglec-8–mediated eosinophil apoptosis. Cytokine priming, which normally prolongs eosinophil survival, paradoxically potentiated this proapoptotic effect. The mechanisms of Siglec-8–mediated apoptosis after priming were therefore explored. Using IL-5 as the priming stimulus, the rate of Siglec-8–induced eosinophil apoptosis was found to be enhanced compared with unprimed cells, and mechanisms differed after IL-5 priming in that neither a pan-caspase inhibitor, nor a specific caspase-3 inhibitor, could override apoptosis. IL-5 priming also accelerated Siglec-8–mediated dissipation of mitochondrial membrane potential. Finally, both the mitochondrial electron transport inhibitor rotenone, and the ROS inhibitors diphenyleneiodonium and antimycin, completely inhibited Siglec-8–mediated apoptosis, even after IL-5 priming. These data demonstrate that IL-5 priming enhances Siglec-8–mediated mitochondrial and ROS-dependent eosinophil apoptosis and eliminates caspase dependence. The potential clinical implication of these findings is that cytokine priming, as often occurs in vivo in asthma and other hypereosinophilic disorders, may render eosinophils from such patients especially susceptible to the proapoptotic effects of a Siglec-8–engaging therapeutic agent. PMID:17690326

  17. NMR studies of two spliced leader RNAs using isotope labeling

    SciTech Connect

    Lapham, J.; Crothers, D.M.

    1994-12-01

    Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions between the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.

  18. A noncoding melanophilin gene (MLPH) SNP at the splice donor of exon 1 represents a candidate causal mutation for coat color dilution in dogs.

    PubMed

    Drögemüller, Cord; Philipp, Ute; Haase, Bianca; Günzel-Apel, Anne-Rose; Leeb, Tosso

    2007-01-01

    Coat color dilution in several breeds of dog is characterized by a specific pigmentation phenotype and sometimes accompanied by hair loss and recurrent skin inflammation, the so-called color dilution alopecia or black hair follicular dysplasia. Coat color dilution (d) is inherited as a Mendelian autosomal recessive trait. In a previous study, MLPH polymorphisms showed perfect cosegregation with the dilute phenotype within breeds. However, different dilute haplotypes were found in different breeds, and no single polymorphism was identified in the coding sequence that was likely to be causative for the dilute phenotype. We resequenced the 5'-region of the canine MLPH gene and identified a strong candidate single nucleotide polymorphism within the nontranslated exon 1, which showed perfect association to the dilute phenotype in 65 dilute dogs from 7 different breeds. The A/G polymorphism is located at the last nucleotide of exon 1 and the mutant A-allele is predicted to reduce splicing efficiency 8-fold. An MLPH mRNA expression study using quantitative reverse transcriptase-polymerase chain reaction confirmed that dd animals had only about approximately 25% of the MLPH transcript compared with DD animals. These results provide preliminary evidence that the reported regulatory MLPH mutation might represent a causal mutation for coat color dilution in dogs. PMID:17519392

  19. Modulation of 2{prime}-5{prime} oligoadenylate synthetase by environmental stress in the marine sponge Geodia cydonium

    SciTech Connect

    Schroeder, H.C.; Wiens, M.; Mueller, W.E.G.; Kuusksalu, A.; Kelve, M.

    1997-07-01

    Recently the authors established the presence of relatively high amounts of 2{prime}-5{prime} oligoadenylates (2{prime}-5{prime} A) and 2{prime}-5{prime} oligoadenylate synthetase (2{prime}-5{prime} A synthetase) in the marine sponge Geodia cydonium. Here they determined by applying radioimmunoassay and high-performance liquid chromatographical methods that the concentration of 2{prime}-5{prime} A synthetase change following exposure of G. cydonium tissue to environmental stress. The 2{prime}-5{prime} A content and the activity of 2{prime}-5{prime} A synthetase, present in crude sponge extract, increase by up to three-fold after treating sponge cubes for 2 h with natural stressors including heat shock (26 C), cold shock (6 C), pH shock (pH 6), and hypertonic shock and subsequent incubation for 18 h under ambient conditions (16 C). No response was observed after exposure of sponges to an alkaline (pH 10) or hypotonic environment. Similar changes have been found for the expression of heat shock protein HSP70 in G. cydonium. These results show that 2{prime}-5{prime} A in sponges may be useful as a novel biomarker for environmental monitoring.

  20. Genomic organization and 5{prime}-flanking DNA sequence of the murine stomatin gene (Epb72)

    SciTech Connect

    Gallagher, P.G.; Turetsky, T.; Mentzer, W.C.

    1996-06-15

    Stomatin is a poorly understood integral membrane protein that is absent from the erythrocyte membranes of many patients with hereditary stomatocytosis. This report describes the cloning of the murine stomatin chromosomal gene, determination of its genomic structure, and characterization of the 5{prime}-flanking genomic DNA sequences. The stomatin gene is encoded by seven exons spread over {approximately}25 kb of genomic DNA. There is no concordance between the exon structure of the stomatin gene and the locations of three domains predicted on the basis of protein structure. Inspection of the 5{prime}-flanking DNA sequences reveals features of a TATA-less housekeeping gene promoter and consensus sequences for a number of potential DNA-binding proteins. 12 refs., 2 figs., 1 tab.

  1. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.

    PubMed Central

    Chillón, M; Dörk, T; Casals, T; Giménez, J; Fonknechten, N; Will, K; Ramos, D; Nunes, V; Estivill, X

    1995-01-01

    mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. Images Figure 3 PMID:7534040

  2. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype

    SciTech Connect

    Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X.; Doerk, T.; Will, K.; Fonknechten, N.

    1995-03-01

    mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.

  3. Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers.

    PubMed Central

    Menting, JGT.; Scopes, R. K.; Stevenson, T. W.

    1994-01-01

    We have detected a flavonoid 3[prime],5[prime]-hydroxylase (F3[prime],5[prime]H) in the microsomal fraction of Petunia hybrida flowers. Activity varied with the development of flowers, peaking immediately prior to and during anthesis, but was absent in mature flowers. F3[prime],5[prime]H activity in flower extracts from genetically defined floral color mutants correlated strictly with the genotypes Hf1 and Hf2. No activity was detected in flowers from mutants homozygous recessive for both alleles. F3[prime],5[prime]H activity was dependent on NADPH and molecular oxygen; there was only slight activity with NADH. The enzyme catalyzes the hydroxylation of 5,7,4[prime]-trihydroxyflavonone at the 3[prime] and 5[prime] positions, and of 5,7,3[prime],4[prime]-tetrahydroxyflavonone and dihydroquercetin at the 5[prime] position. Hydroxylase activity was inhibited by plant growth regulators (1-aminobenzotriazole and tetcyclacis) and by CO, N-ethylmaleimide, diethyldithiocarbamate, and cytochrome (Cyt) c. Activity was not affected by diethylpyrocarbonate or phenylmethylsulfonyl fluoride, but was enhanced by 2-mercaptoethanol. A polyclonal antibody that inhibits higher plant NADPH-Cyt P450 reductase inhibited the F3[prime],5[prime]H. The data are consistent with the suggestion that the P. hybrida F3[prime],5[prime]H is a monooxygenase consisting of a Cyt P450 and a NADPH-Cyt P-450 reductase. Cyts P450 were detected in microsomal membranes and in solubilized detergent extracts of these membranes. F3[prime],5[prime]H activity was sensitive to low concentrations of all detergents tested, and therefore solubilization of the active enzyme was not achieved. Reaction products other than flavanones were observed in F3[prime],5[prime]H assays and these may be formed by enzymic oxidation of flavanones. The possibility of a microsomal flavone synthase of a type that has not been described in P. hybrida is discussed. PMID:12232356

  4. 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. PMID:25720307

  5. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  6. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  7. Splicing Programs and Cancer

    PubMed Central

    Germann, Sophie; Gratadou, Lise; Dutertre, Martin; Auboeuf, Didier

    2012-01-01

    Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets. PMID:22132318

  8. Expression of splice variants of mts1 gene in normal and neoplastic human tissues

    SciTech Connect

    Ambartsumyan, N.S. |; Grigorian, M.S.; Lukanidin, E.M.

    1995-09-01

    Data on cloning of cDNA corresponding to human mts1 gene transcripts are presented. By comparing nucleotide sequences of the genomic DNA clone and cDNA of mts1, it was shown that human osteosarcoma OHS cells contain two alternative splice variants of mts1 transcripts. Alternative splicing occurs in the 5{prime}-untranslated region of the mts1 pre-mRNA. Both splice variants, hu-mts1 and hu-mts1(var), demonstrate similar stability in the cells, and each contains one open reading frame for the MTS1 protein. However, the two types of transcripts are translated with different effectiveness. The level of transcription of mts1 splice variants in different normal and neoplastic tissues and cell lines varies significantly. The role of alternative splicing as the mechanism responsible for posttranscriptional regulation of mts1 gene expression is discussed. 31 refs., 5 figs.

  9. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  10. An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

    PubMed Central

    Pastor, Tibor; Talotti, Gabriele; Lewandowska, Marzena Anna; Pagani, Franco

    2009-01-01

    We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations. PMID:19773425

  11. Probabilistic simple splicing systems

    NASA Astrophysics Data System (ADS)

    Selvarajoo, Mathuri; Heng, Fong Wan; Sarmin, Nor Haniza; Turaev, Sherzod

    2014-06-01

    A splicing system, one of the early theoretical models for DNA computing was introduced by Head in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings of DNA molecules at the specific recognition sites and attaches the prefix of the first string to the suffix of the second string, and the prefix of the second string to the suffix of the first string, thus yielding the new strings. For a specific type of splicing systems, namely the simple splicing systems, the recognition sites are the same for both strings of DNA molecules. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions have been considered for splicing systems in order to increase their computational power. Recently, probabilistic splicing systems have been introduced where the probabilities are initially associated with the axioms, and the probabilities of the generated strings are computed from the probabilities of the initial strings. In this paper, some properties of probabilistic simple splicing systems are investigated. We prove that probabilistic simple splicing systems can also increase the computational power of the splicing languages generated.

  12. Altered PLP1 splicing causes hypomyelination of early myelinating structures

    PubMed Central

    Kevelam, Sietske H; Taube, Jennifer R; van Spaendonk, Rosalina M L; Bertini, Enrico; Sperle, Karen; Tarnopolsky, Mark; Tonduti, Davide; Valente, Enza Maria; Travaglini, Lorena; Sistermans, Erik A; Bernard, Geneviève; Catsman-Berrevoets, Coriene E; van Karnebeek, Clara D M; Østergaard, John R; Friederich, Richard L; Fawzi Elsaid, Mahmoud; Schieving, Jolanda H; Tarailo-Graovac, Maja; Orcesi, Simona; Steenweg, Marjan E; van Berkel, Carola G M; Waisfisz, Quinten; Abbink, Truus E M; van der Knaap, Marjo S; Hobson, Grace M; Wolf, Nicole I

    2015-01-01

    Objective The objective of this study was to investigate the genetic etiology of the X-linked disorder “Hypomyelination of Early Myelinating Structures” (HEMS). Methods We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients’ fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus–Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing. PMID:26125040

  13. RNA polymerase II pauses at the 5 prime end of the transcriptionally induced Drosophila hsp70 gene

    SciTech Connect

    O'Brien, T.; Lis, J.T. )

    1991-10-01

    An RNA polymerase II molecule is associated with the 5{prime} end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides. Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here the authors report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5{prime} end of hsp70 was transcribed approximately five times during the 25-min heat shock that they used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

  14. Binding of nickel /II/ to 5-prime-nucleoside monophosphates and related compounds. [role in origin of life

    NASA Technical Reports Server (NTRS)

    Orenberg, J. B.; Kjos, K. M.; Winkler, R.; Link, J.; Lawless, J. G.

    1982-01-01

    The interactions of Ni(II) cation with a representative suite of purine bases and the respective nucleosides and nucleotides have been studied by ultraviolet difference spectroscopy. Apparent association constants were determined for each system at pH 7.0, using computer linear regression coupled with an iteration technique. The specificity of binding of Ni(2+) for the purine nucleotides studied at pH 7.0 was 5-prime-GMP greater than 5-prime-AMP; a similar ordering was also found for the respective nucleosides and bases. In this study binding was not observed for the suite of pyramidines used, although an Ni(2+) -cytidine complex has been observed (Fiskin and Beer, 1965). It was also found that Ni(2+) bound more strongly to the purine 5-prime-nucleotides than to the respective nucleosides and bases. These trends are explained in terms of metal-ligand bonds and available bonding positions on the ligands. A role for metal-ion-nucleotide types of complexes is suggested in the processes that might have given rise to the origin of life.

  15. Soluble minerals in chemical evolution. I - Adsorption of 5-prime-AMP on CaSO4 - A model system

    NASA Technical Reports Server (NTRS)

    Orenberg, J. B.; Chan, S.; Calderon, J.; Lahav, N.

    1985-01-01

    The adsorption of 5-prime-AMP onto solid CaSO4-2H2O was studied in a saturated suspension as a function of pH and electrolyte concentration. The adsorption is pH-dependent and is directly correlated with the charge on the 5-prime-AMP molecule which is determined by the state of protonation of the N-1 nitrogen of the purine ring and the phosphate oxygens. It is proposed that the binding that occurs between the nucleotide and the salt is electrostatic in nature. The adsorption decreases with increasing ionic strength of the solution which means that in a fluctuating environment of wetting and drying cycles, a biomolecule similar to 5-prime-AMP could be expected to desorb during the drying phase. The results indicate that CaSO4-2H2O can serve as a concentrating surface for biomolecules. The significance of this is discussed with regard to the possible role of soluble minerals and their surfaces in a geochemical model consistent with the evolution of the earth and the origin of life.

  16. Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

    PubMed Central

    Kiss-László, Z; Blanc, S; Hohn, T

    1995-01-01

    A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV. Images PMID:7628455

  17. The evolution of spliced leader trans-splicing in nematodes.

    PubMed

    Pettitt, Jonathan; Harrison, Neale; Stansfield, Ian; Connolly, Bernadette; Müller, Berndt

    2010-08-01

    Spliced leader trans-splicing occurs in many primitive eukaryotes including nematodes. Most of our knowledge of trans-splicing in nematodes stems from the model organism Caenorhabditis elegans and relatives, and from work with Ascaris. Our investigation of spliced leader trans-splicing in distantly related Dorylaimia nematodes indicates that spliced-leader trans-splicing arose before the nematode phylum and suggests that the spliced leader RNA gene complements in extant nematodes have evolved from a common ancestor with a diverse set of spliced leader RNA genes. PMID:20659016

  18. Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences.

    PubMed Central

    Teraoka, S N; Telatar, M; Becker-Catania, S; Liang, T; Onengüt, S; Tolun, A; Chessa, L; Sanal, O; Bernatowska, E; Gatti, R A; Concannon, P

    1999-01-01

    Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations. PMID:10330348

  19. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene

    SciTech Connect

    Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. Erasmus Univ. of Rotterdam )

    1993-03-01

    The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. The authors studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene [alpha], along with their 5[prime]- and 3[prime]-flanking regions, the authors identified a 4-base deletion at the 3[prime]-boundary of exon 6 in one GR allele ([Delta][sub 4]), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G[sub 1220]) in the proband, in one of her affected brothers and in her unaffected sister. This deletion in the glucocorticoid receptor gene was associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism. 40 refs., 3 figs.

  20. Alternative Splicing in CKD.

    PubMed

    Stevens, Megan; Oltean, Sebastian

    2016-06-01

    Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit. PMID:26763787

  1. The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

    PubMed Central

    Emmert, Steffen; Schneider, Thomas D.; Khan, Sikandar G.; Kraemer, Kenneth H.

    2001-01-01

    Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP–Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA. PMID:11266544

  2. A five prime splice-region G yields C mutation in exon 1 of the human. beta. -globin gene inhibits pre-mRNA splicing: A mechanism for. beta. sup + -thalassemia

    SciTech Connect

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M. ); Gattoni, R.; Stevenin, J. ); Chibani, J. )

    1989-02-01

    The authors have characterized a Mediterranean {beta}-thalassemia allele containing a sequence change at codon 30 that alters both {beta}-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G {yields} C transversion at position {minus}1 of intron 1 reduces severely the utilization of the normal 5{prime} splice site since the level of the Arg {yields} Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position {minus}1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5{prime} splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.

  3. Linkage disequilibrium in the insulin gene region: Size variation at the 5{prime} flanking polymorphism and bimodality among {open_quotes}Class I{close_quotes} alleles

    SciTech Connect

    McGinnis, R.E.; Spielman, R.S.

    1994-09-01

    The 5{prime} flanking polymorphism (5{prime}FP), a hypervariable region at the 5{prime} end of the insulin gene, has {open_quotes}class 1{close_quotes} alleles (650-900 bp long) that are in positive linkage disequilibrium with insulin-dependent diabetes mellitus (IDDM). The authors report that precise sizing of the 5{prime}FP yields a bimodal frequency distribution of class 1 allele lengths. Class 1 alleles belonging to the lower component (650-750 bp) of the bimodal distribution were somewhat more highly associated with IDDM than were alleles from the upper component (760-900 bp), but the difference was not statistically significant. They also examined 5{prime}FP length variation in relation to allelic variation at nearby polymorphisms. At biallelic RFLPs on both sides of the 5{prime}FP, they found that one allele exhibits near-total association with the upper component of the 5FP class 1 distribution. Such associations represent a little-known but potentially wide-spread form of linkage disequilibrium. In this type of disequilibrium, a flanking allele has near-complete association with a single mode of VNTR alleles whose lengths represent consecutive numbers of tandem repeats (CNTR). Such extreme disequilibrium between a CNTR mode and flanking alleles may originate and persist because length mutations at some VNTR loci usually add or delete only one or two repeat units. 22 refs., 5 figs., 6 tabs.

  4. Constant splice-isoform ratios in human lymphoblastoid cells support the concept of a splico-stat.

    PubMed

    Kramer, Marcel; Huse, Klaus; Menzel, Uwe; Backhaus, Oliver; Rosenstiel, Philip; Schreiber, Stefan; Hampe, Jochen; Platzer, Matthias

    2011-03-01

    Splicing generates mature transcripts from genes in pieces in eukaryotic cells. Overwhelming evidence has accumulated that alternative routes in splicing are possible for most human and mammalian genes, thereby allowing formation of different transcripts from one gene. No function has been assigned to the majority of identified alternative splice forms, and it has been assumed that they compose inert or tolerated waste from aberrant or noisy splicing. Here we demonstrate that five human transcription units (WT1, NOD2, GNAS, RABL2A, RABL2B) have constant splice-isoform ratios in genetically diverse lymphoblastoid cell lines independent of the type of alternative splicing (exon skipping, alternative donor/acceptor, tandem splice sites) and gene expression level. Even splice events that create premature stop codons and potentially trigger nonsense-mediated mRNA decay are found at constant fractions. The analyzed alternative splicing events were qualitatively but not quantitatively conserved in corresponding chimpanzee cell lines. Additionally, subtle splicing at tandem acceptor splice sites (GNAS, RABL2A/B) was highly constrained and strongly depends on the upstream donor sequence content. These results also demonstrate that unusual and unproductive splice variants are produced in a regulated manner. PMID:21220357

  5. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    PubMed Central

    Gérard, Xavier; Perrault, Isabelle; Munnich, Arnold; Kaplan, Josseline; Rozet, Jean-Michel

    2015-01-01

    Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15%) creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO) allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2'-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA)-approved adeno-associated virus (AAV)-vectors, thus hampering gene augmentation therapy. PMID:26325627

  6. The Peculiarities of Large Intron Splicing in Animals

    PubMed Central

    Fedorov, Alexei

    2009-01-01

    In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These “large introns” must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5′ and 3′ acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing—a consecutive splicing from the 5′-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs) that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites. PMID:19924226

  7. Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

    PubMed

    Straniero, Letizia; Rimoldi, Valeria; Soldà, Giulia; Mauri, Lucia; Manfredini, Emanuela; Andreucci, Elena; Bargiacchi, Sara; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Asselta, Rosanna; Primignani, Paola

    2015-09-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient. PMID:26016411

  8. Electrical-splicing connector

    NASA Technical Reports Server (NTRS)

    Stringer, E. J.

    1977-01-01

    Connection can be made without removing insulation, and connector case insulates splice. Device can be made in various sizes and saves time, especially when working on prototype boards with several interconnecting test leads.

  9. The missing puzzle piece: splicing mutations

    PubMed Central

    Lewandowska, Marzena A

    2013-01-01

    Proper gene splicing is highly dependent on the correct recognition of exons. Among the elements allowing this process are the “cis” (conserved sequences) and “trans” (snRNP, splicing factors) elements. Splicing mutations are related with a number of genetic disorders and usually induce exon skipping, form new exon/intron boundaries or activate new cryptic exons as a result of alterations at donor/acceptor sites. They constitute more than 9% of the currently published mutations, but this value is highly underestimated as many of the potential mutations are located in the “cis” elements and should be confirmed experimentally. The most commonly detected splicing mutations are located at donor (5’) and acceptor (3’) sites. Mutations at the branch point are rare (only over a dozen are known to date), and are mostly searched and detected when no alteration has been detected in the sequenced exons and UTRs. Polypyrimidine tract mutations are equally rare. High throughput technologies, as well as traditional Sanger sequencing, allow detection of many changes in intronic sequences and intron/exon boundaries. However, the assessment whether a mutation affects exon recognition and results in a genetic disorder has to be conducted using molecular biology methods: in vitro transcription of the sequence of interest cloned into a plasmid, with and without alterations, or mutation analysis via a hybrid minigene system. Even though microarrays and new generation sequencing methods pose difficulties in detecting novel branch point mutations, these tools seem appropriate to expand the mutation detection panel especially for diagnostic purposes. PMID:24294354

  10. pH profile of the adsorption of nucleotides onto montmorillonite. II - Adsorption and desorption of 5-prime-AMP in iron-calcium montmorillonite systems

    NASA Technical Reports Server (NTRS)

    Banin, A.; Lawless, J. G.; Mazzurco, J.; Church, F. M.; Margulies, L.; Orenberg, J. B.

    1985-01-01

    The interaction of 5-prime-AMP with montmorillonite saturated with various ratios of two metals found ubiquitously on the surface of earth, that is, iron and calcium, is investigated. Adsorption and desorption of the nucleotide were studied in the pH range of 2-12 at three levels of addition: 0.080, 0.268 and 0.803 mmole 5-prime-AMP per gram of clay. Two desorption stages were employed - H2O wash and NaOH extraction (pH = 12.0). 5-prime-AMP was preferentially adsorbed on the Fe-containing clays relative to the Ca clay. The nucleotide was fully recovered by the two desorption stages, mostly by the NaOH extraction. The evidence at hand indicates that 5-prime-AMP reaction with clay is affected by electrostatic interactions involving both attraction and repulsion forces. Some specific adsorption, possibly the result of covalent bonding and complex formation with the adsorbed ion, cannot be ruled out for iron but does not appear to operate for calcium. Changes in pH cause varying degrees of attaction and repulsion of 5-prime-AMP and may have been operating on the primitive earth, leading to sequences of adsorption and release of this biomolecule.

  11. Linkage disequilibrium in the insulin gene region is related to the exact number of repeat units present at the 5{prime} flanking polymorphism

    SciTech Connect

    McGinnis, R.E.; Spielman, R.S.

    1994-09-01

    Tandem DNA repeat units (RUs) located 5{prime} to the insulin (INS) gene give rise to a {open_quotes}5{prime} flanking polymorphism{close_quotes} (5{prime}FP) with minisatellite alleles belonging to 3 size classes. The shortest or {open_quotes}class 1{close_quotes} alleles (mean length of {approximately}40 RUs) are associated with insulin-dependent diabetes mellitus (IDDM), and the 5{prime}FP is one of several INS region loci in strong linkage disequilibrium with IDDM. We have amplified class 1 alleles and have determined the exact number of RUs in individual class 1 alleles found in parents of 50 IDDM families. We also obtained INS region haplotypes by typing two loci near tyrosine hydroxylase (TH) and two loci near insulin-like growth factor II (IGF2). We obtained these results: (1) Class 1 alleles (n=101) were found at every integer length from 30 to 44 RUs, the lengths of smallest and largest class 1 alleles observed. The allele frequency distribution was trimodal with peaks at 31, 40 and 42 RUs; 18%, 34% and 48% of the alleles belonged to the three components, respectively. (2) Allelic variation at each flanking locus was highly associated with the exact number of RUs present at the 5{prime}FP. Our results suggest that creation of new 5{prime}FP or other minisatellite haplotypes may be {open_quotes}constrained{close_quotes} in that flanking alleles usually become associated with a new minisatellite length different by only one or two RUs. Furthermore, since many flanking alleles were associated with a single narrow range of class 1 integer lengths, determining exact RU length may aid in visualizing linkage disequilibrium and allelic associations involving other minisatellite loci.

  12. Splice junctions in adenovirus 2 early region 4 mRNAs: multiple splice sites produce 18 to 24 RNAs.

    PubMed Central

    Tigges, M A; Raskas, H J

    1984-01-01

    We localized the splice junctions in adenovirus 2 early region 4 (E4) mRNAs. Processing of the E4 precursor RNA positioned the donor splice site of the 5' leader sequence adjacent to acceptor sites near the 5' ends of five of the six open reading regions in the E4 transcription unit. Of particular interest among the E4 mRNAs is an extensively spliced class which includes multiple species with sizes ranging from 1.1 to 0.75 kilobases (kb). Purified 1.1- to 0.75-kb mRNAs specified at least 10 polypeptides in vitro. We detected eight acceptor and two donor splice sites utilized in the deletion of the intron from the 3' portion of these mRNAs. E4 RNAs were isolated from the cytoplasm of infected cells at 5, 9, 12, and 18 h after infection. The E4 mRNAs were present throughout infection, but different members of the 1.1- to 0.7-kb class were predominant at each time assayed. Alternate splicing of the 3.0-kb E4 precursor RNA can generate as many as 25 mRNAs that encode at least 16 polypeptides. Images PMID:6336328

  13. Extracellular matrix and hormones transcriptionally regulate bovine. beta. -casein 5 prime sequences in stably transfected mouse mammary cells

    SciTech Connect

    Schmidhauser, C. Bissell, M.J. ); Myers, C.A.; Casperson, G.F. )

    1990-12-01

    Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

  14. Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders.

    PubMed

    Pettitt, Jonathan; Müller, Berndt; Stansfield, Ian; Connolly, Bernadette

    2008-04-01

    The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda. PMID:18256244

  15. Splice assembly tool and method of splicing

    DOEpatents

    Silva, Frank A.

    1980-01-01

    A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.

  16. Rapid generation of splicing reporters with pSpliceExpress

    PubMed Central

    Kishore, Shivendra; Khanna, Amit; Stamm, Stefan

    2008-01-01

    Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo. PMID:18930792

  17. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.)

    PubMed Central

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef

    2010-01-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  18. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    PubMed

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  19. BUILDING ROBUST TRANSCRIPTOMES WITH MASTER SPLICING FACTORS

    PubMed Central

    Jangi, Mohini; Sharp, Phillip A.

    2014-01-01

    Coherent splicing networks arise from many discrete splicing decisions regulated in unison. Here, we examine the properties of robust, context-specific splicing networks. We propose that a subset of key splicing regulators, or “master splicing factors,” respond to environmental cues to establish and maintain tissue transcriptomes during development. PMID:25417102

  20. Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5 prime -triphosphate

    SciTech Connect

    Vollmer, S.H.; Colman, R.F. )

    1990-03-13

    The affinity label 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5{prime}-triphosphate (8-BDB-TA-5{prime}-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K{sup +}, and Mn{sup 2+} and only 1 mol of reagent/mol of subunit is incorporated. The authors have now identified the resultant modified residues. After reaction with 8-BDB-TA-5{prime}-TP at pH 7.0, modified enzyme was incubated with ({sup 3}H)NaBH{sub 4} to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5{prime}-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn{sup 162}-Ile-Cys-Lys{sup 165} and Cys{sup 151}-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys{sup 161}, with a smaller amount of Asn{sup 43}-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg{sup 55}. Reaction in the presence of the protectants phosphoenolpyruvate, K{sup +}, and Mn{sup 2+} yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys{sup 151} and Cys{sup 48}.

  1. Accuracy of Deoxynucleotide Incorporation by Soybean Chloroplast DNA Polymerases Is Independent of the Presence of a 3[prime] to 5[prime] Exonuclease.

    PubMed Central

    Bailey, J. C.; Heinhorst, S.; Cannon, G. C.

    1995-01-01

    DNA polymerase was purified from soybean (Glycine max) chloroplasts that were actively replicating DNA. The main form (form I) of the enzyme was associated with a low level of 3[prime] to 5[prime] exonuclease activity throughout purification, although the ratio of exonuclease to polymerase activity decreased with each successive purification step. A second form (form II) of DNA polymerase, which elutes from DEAE-cellulose at a higher salt concentration than form I, was devoid of any exonuclease activity. To assess the potential function of the 3[prime] to 5[prime] exonuclease in proofreading, the fidelity of deoxynucleotide incorporation was measured for form I DNA polymerase throughout purification. Despite the steadily decreasing ratio of 3[prime] to 5[prime] exonuclease to polymerase activity, the extent of misincorporation by form I enzyme remained unchanged during the final purification steps, suggesting that the exonuclease did not contribute to the accuracy of DNA synthesis by this polymerase. Fidelity of form I DNA polymerase, when compared with that of form II, revealed a higher level of misincorporation for form I enzyme, a finding that is consistent with the exonuclease playing little or no role in exonucleolytic proofreading. PMID:12228434

  2. Splicing Wires Permanently With Explosives

    NASA Technical Reports Server (NTRS)

    Bement, Laurence J.; Kushnick, Anne C.

    1990-01-01

    Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.

  3. Alternative RNA splicing and cancer

    PubMed Central

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  4. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    SciTech Connect

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  5. ERISdb: a database of plant splice sites and splicing signals.

    PubMed

    Szcześniak, Michał Wojciech; Kabza, Michał; Pokrzywa, Rafał; Gudyś, Adam; Makałowska, Izabela

    2013-02-01

    Splicing is one of the major contributors to observed spatiotemporal diversification of transcripts and proteins in metazoans. There are numerous factors that affect the process, but splice sites themselves along with the adjacent splicing signals are critical here. Unfortunately, there is still little known about splicing in plants and, consequently, further research in some fields of plant molecular biology will encounter difficulties. Keeping this in mind, we performed a large-scale analysis of splice sites in eight plant species, using novel algorithms and tools developed by us. The analyses included identification of orthologous splice sites, polypyrimidine tracts and branch sites. Additionally we identified putative intronic and exonic cis-regulatory motifs, U12 introns as well as splice sites in 45 microRNA genes in five plant species. We also provide experimental evidence for plant splice sites in the form of expressed sequence tag and RNA-Seq data. All the data are stored in a novel database called ERISdb and are freely available at http://lemur.amu.edu.pl/share/ERISdb/. PMID:23299413

  6. Alternative splicing and muscular dystrophy

    PubMed Central

    Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

    2013-01-01

    Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle. PMID:20603608

  7. Therapeutic targeting of splicing in cancer.

    PubMed

    Lee, Stanley Chun-Wei; Abdel-Wahab, Omar

    2016-09-01

    Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein-RNA interactions in cancer. PMID:27603132

  8. Tissue-specific methylation of individual CpG dinucleotides in the 5{prime} upstream region of the mouse catalase gene (Cas-1)

    SciTech Connect

    Pillay, I.L.; Singh, S.M.

    1994-09-01

    The intracellular antioxidant enzyme, catalase, is encoded by a gene whose level of expression in different organisms, including humans, varies with tissue-type. The {open_quotes}TATA-less{close_quotes} 5{prime} upstream region of the catalase gene, in mice and humans, contains a CpG island. Such CG-rich regions are target sites for cytosine methylation and have been implicated in tissue-specific gene expression. However, the methylation status of individual CpG dinucleotides and their significance in gene expression has not been established. A 275 bp fragment within the 5{prime} region of Cas-1 was evaluated for CpG methylation. HpaII digestion of genomic DNA, followed by polymerase chain reaction amplification (HpaII-PCR), suggests that at least one of three CCGG is not methylated in nine different somatic tissues that express this enzyme at various levels. In contrast, all three CCGG sites are methylated in DNA from sperm and spleen. Further examination of the methylation specificity of individual CCGG sites was conducted using sodium bisulfite modification of genomic DNA followed by HPaII-PCR. Sodium bisulfite modifies non-methylated cytosines to uracils, changing a CG to a TG dinucleotide. This nucleotide substitution eliminates HpaII sites and allows the methylation status of each of the CCGG sites to be assessed. The ability to discern the number and combination of methylated sites within the 5{prime} region of a gene permits the determination of a possible correlation between differential methylation patterns and temporal/spatial gene regulation. Analysis of differential methylation, using the mouse catalase gene as a model, provides further insight into CpG methylation as one mechanism of mammalian gene regulation.

  9. Nucleotide sequence composition adjacent to intronic splice sites improves splicing efficiency via its effect on pre-mRNA local folding in fungi.

    PubMed

    Zafrir, Zohar; Tuller, Tamir

    2015-10-01

    RNA splicing is the central process of intron removal in eukaryotes known to regulate various cellular functions such as growth, development, and response to external signals. The canonical sequences indicating the splicing sites needed for intronic boundary recognition are well known. However, the roles and evolution of the local folding of intronic and exonic sequence features adjacent to splice sites has yet to be thoroughly studied. Here, focusing on four fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, and Candida albicans), we performed for the first time a comprehensive high-resolution study aimed at characterizing the encoding of intronic splicing efficiency in pre-mRNA transcripts and its effect on intron evolution. Our analysis supports the conjecture that pre-mRNA local folding strength at intronic boundaries is under selective pressure, as it significantly affects splicing efficiency. Specifically, we show that in the immediate region of 12-30 nucleotides (nt) surrounding the intronic donor site there is a preference for weak pre-mRNA folding; similarly, in the region of 15-33 nt surrounding the acceptor and branch sites there is a preference for weak pre-mRNA folding. We also show that in most cases there is a preference for strong pre-mRNA folding further away from intronic splice sites. In addition, we demonstrate that these signals are not associated with gene-specific functions, and they correlate with splicing efficiency measurements (r = 0.77, P = 2.98 × 10(-21)) and with expression levels of the corresponding genes (P = 1.24 × 10(-19)). We suggest that pre-mRNA folding strength in the above-mentioned regions has a direct effect on splicing efficiency by improving the recognition of intronic boundaries. These new discoveries are contributory steps toward a broader understanding of splicing regulation and intronic/transcript evolution. PMID:26246046

  10. A new family of retroviral long terminal repeat elements in the human genome identified by their homologies to an element 5{prime} to the spider monkey haptoglobin gene

    SciTech Connect

    Erickson, L.M.; Maeda, N.

    1995-06-10

    A new family of retroviral long terminal repeats that we name Spm-LTR has been identified as a result of DNA sequence comparisons between the entire Gen-Bank databank and an element, SPHP, located 5{prime} to the haptoglobin gene of spider monkeys. The 18 human Spm-LTR sequences so identified fall into three subtypes. There is no sequence similarity between Spm-LTR elements and any endogenous retroviral LTR sequences previously reported except for general features that define LTRs. However, a previously described repeated sequence (MER-4) forms a portion of the Spm-LTR sequence. 13 refs., 1 fig., 1 tab.

  11. Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

    PubMed

    O'Neill, J P; Rogan, P K; Cariello, N; Nicklas, J A

    1998-11-01

    The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are

  12. Minimum Factorization Agreement of Spliced ESTs

    NASA Astrophysics Data System (ADS)

    Bonizzoni, Paola; Della Vedova, Gianluca; Dondi, Riccardo; Pirola, Yuri; Rizzi, Raffaella

    Producing spliced EST sequences is a fundamental task in the computational problem of reconstructing splice and transcript variants, a crucial step in the alternative splicing investigation. Now, given an EST sequence, there can be several spliced EST sequences associated to it, since the original EST sequences may have different alignments against wide genomic regions.

  13. A novel computational method for the identification of plant alternative splice sites.

    PubMed

    Cui, Ying; Han, Jiuqiang; Zhong, Dexing; Liu, Ruiling

    2013-02-01

    Alternative splicing (AS) increases protein diversity by generating multiple transcript isoforms from a single gene in higher eukaryotes. Up to 48% of plant genes exhibit alternative splicing, which has proven to be involved in some important plant functions such as the stress response. A hybrid feature extraction approach which combing the position weight matrix (PWM) with the increment of diversity (ID) was proposed to represent the base conservative level (BCL) near splice sites and the similarity level of two datasets, respectively. Using the extracted features, the support vector machine (SVM) was applied to classify alternative and constitutive splice sites. By the proposed algorithm, 80.8% of donor sites and 85.4% of acceptor sites were correctly classified. It is anticipated that the novel computational method is promising for the identification of AS sites in plants. PMID:23313482

  14. Functional correction by antisense therapy of a splicing mutation in the GALT gene.

    PubMed

    Coelho, Ana I; Lourenço, Sílvia; Trabuco, Matilde; Silva, Maria João; Oliveira, Anabela; Gaspar, Ana; Diogo, Luísa; Tavares de Almeida, Isabel; Vicente, João B; Rivera, Isabel

    2015-04-01

    In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314

  15. Biological effects of exogenous adenosine 5 prime -triphosphate on cultured mammalian cells: Evidence for a receptor mechanism and its regulation by desensitization

    SciTech Connect

    Gonzalez, F.A.

    1989-01-01

    Exogenous adenosine 5{prime}-triphosphate (ATP) mobilized intracellular calcium in human carcinoma A43l cells and in Swiss 3T3 and 3T6 mouse fibroblasts by increasing inositol trisphosphate similar to well down growth factors (platelet-derived growth factor (PDGF), epidermal growth factor (EGF), bradykinin (BK), serum). Calcium mobilization was examined by video imaging of fura-2 fluorescence is single cells, following the radioactive isotope {sup 45}Ca, and monitoring the decrease in fluorescence of cells loaded with chlortetracycline. Uridine 5{prime}-triphosphate, but not other nucleotides, mimicked ATP. Single-cell analysis revealed synchronous responses in 10 sec to ATP, BK or serum, while PDGF (3T3) and EGF (A431) produced slower signals with significant cell-to-cell variation. PDGF desensitized 3T3 cells to ATP and BK added 100 sec later but ATP or BK did not desensitized to PDGF. Homologous desensitization was seen with all agonists. Heterologous desensitization was also observed in A431 cells where ATP desensitized to serum, but serum did not to ATP. ATP-stimulated calcium entry was detected after 10 sec in A431 cells, but not in Swiss 3T6 cells. Entry started before significant efflux had occurred and did not fit the capacitance model of Putney. A 2-3 hr ATP pretreatment produced a homologous desensitization state that required 20 hr to disappear, probably due to down-regulation of the putative ATP receptors.

  16. Synthesis and characterization of ( sup 3 H)-5 prime azido-N-1-naphthylphthlamic acid, a photolabile N-1-naphthylphthalamic acid analog

    SciTech Connect

    Voet, J.G.; Dodge, B.; Harris, K.; Jacobs, M.; Larkin, L.; Bader, S.; Schnitzler, G.; Sutherland, J. )

    1990-05-01

    The NPA (N-1-naphthylphthalamic acid) receptor is an important protein involved in the regulation of transport of indole-3-acetic acid (IAA). In our attempt to isolate and characterize this protein we have previously synthesized and characterized a photolabile analog of NPA, 5{prime}-azido-NPA (Az-NPA) and shown it to be a competitor of NPA for binding sites on the NPA receptor as well as an inhibitor of auxin transport. We have now synthesized and characterized ({sup 3}H)-Az-NPA. The precursor, 2,3,4,5-Br-5{prime}-amino-NPA was dehydrohalogenated with tritium gas by Research Products International. The amino group was converted to an azido group and the product purified by HPLC. ({sup 3}H)-Az-NPA was found to be photolabile and to co-chromatograph with our synthetic unlabeled Az-NPA. Furthermore, the tritiated material was found to bind to zucchini hypocotyl cell membranes in a manner competitive with NPA as well as unlabeled Az-NPA. Photolysis of zucchini phase-partitioned plasma membranes in the presence of ({sup 3}H)-Az-NPA resulted in covalent association of tritium with the membranes. Much of this covalent association could be prevented by prior treatment of the membranes with excess NPA.

  17. Splicing Efficiently Couples Optical Fibers

    NASA Technical Reports Server (NTRS)

    Lutes, G. F.

    1985-01-01

    Method of splicing single-mode optical fibers results in very low transmission losses through joined fiber ends. Coupling losses between joined optical-fiber ends only 0.1 dB. Method needs no special operator training.

  18. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

    PubMed Central

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  19. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  20. SpliceVista, a Tool for Splice Variant Identification and Visualization in Shotgun Proteomics Data*

    PubMed Central

    Zhu, Yafeng; Hultin-Rosenberg, Lina; Forshed, Jenny; Branca, Rui M. M.; Orre, Lukas M.; Lehtiö, Janne

    2014-01-01

    Alternative splicing is a pervasive process in eukaryotic organisms. More than 90% of human genes have alternatively spliced products, and aberrant splicing has been shown to be associated with many diseases. Current methods employed in the detection of splice variants include prediction by clustering of expressed sequence tags, exon microarray, and mRNA sequencing, all methods focusing on RNA-level information. There is a lack of tools for analyzing splice variants at the protein level. Here, we present SpliceVista, a tool for splice variant identification and visualization based on mass spectrometry proteomics data. SpliceVista retrieves gene structure and translated sequences from alternative splicing databases and maps MS-identified peptides to splice variants. The visualization module plots the exon composition of each splice variant and aligns identified peptides with transcript positions. If quantitative mass spectrometry data are used, SpliceVista plots the quantitative patterns for each peptide and provides users with the option to cluster peptides based on their quantitative patterns. SpliceVista can identify splice-variant-specific peptides, providing the possibility for variant-specific analysis. The tool was tested on two experimental datasets (PXD000065 and PXD000134). In A431 cells treated with gefitinib, 2983 splice-variant-specific peptides corresponding to 939 splice variants were identified. Through comparison of splice-variant-centric, protein-centric, and gene-centric quantification, several genes (e.g. EIF4H) were found to have differentially regulated splice variants after gefitinib treatment. The same discrepancy between protein-centric and splice-centric quantification was detected in the other dataset, in which induced pluripotent stem cells were compared with parental fibroblast and human embryotic stem cells. In addition, SpliceVista can be used to visualize novel splice variants inferred from peptide-level evidence. In summary, Splice

  1. SpliceVista, a tool for splice variant identification and visualization in shotgun proteomics data.

    PubMed

    Zhu, Yafeng; Hultin-Rosenberg, Lina; Forshed, Jenny; Branca, Rui M M; Orre, Lukas M; Lehtiö, Janne

    2014-06-01

    Alternative splicing is a pervasive process in eukaryotic organisms. More than 90% of human genes have alternatively spliced products, and aberrant splicing has been shown to be associated with many diseases. Current methods employed in the detection of splice variants include prediction by clustering of expressed sequence tags, exon microarray, and mRNA sequencing, all methods focusing on RNA-level information. There is a lack of tools for analyzing splice variants at the protein level. Here, we present SpliceVista, a tool for splice variant identification and visualization based on mass spectrometry proteomics data. SpliceVista retrieves gene structure and translated sequences from alternative splicing databases and maps MS-identified peptides to splice variants. The visualization module plots the exon composition of each splice variant and aligns identified peptides with transcript positions. If quantitative mass spectrometry data are used, SpliceVista plots the quantitative patterns for each peptide and provides users with the option to cluster peptides based on their quantitative patterns. SpliceVista can identify splice-variant-specific peptides, providing the possibility for variant-specific analysis. The tool was tested on two experimental datasets (PXD000065 and PXD000134). In A431 cells treated with gefitinib, 2983 splice-variant-specific peptides corresponding to 939 splice variants were identified. Through comparison of splice-variant-centric, protein-centric, and gene-centric quantification, several genes (e.g. EIF4H) were found to have differentially regulated splice variants after gefitinib treatment. The same discrepancy between protein-centric and splice-centric quantification was detected in the other dataset, in which induced pluripotent stem cells were compared with parental fibroblast and human embryotic stem cells. In addition, SpliceVista can be used to visualize novel splice variants inferred from peptide-level evidence. In summary, Splice

  2. Spliced-leader trans-splicing in freshwater planarians.

    PubMed

    Zayas, Ricardo M; Bold, Tyler D; Newmark, Phillip A

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing. PMID:15972844

  3. Methods for Characterization of Alternative RNA Splicing

    PubMed Central

    Harvey, Samuel E.; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing “minigene” in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest. PMID:26721495

  4. MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery

    PubMed Central

    Wang, Kai; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J.; Huang, Yan; Savich, Gleb L.; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A.; Perou, Charles M.; MacLeod, James N.; Chiang, Derek Y.; Prins, Jan F.; Liu, Jinze

    2010-01-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice. PMID:20802226

  5. MapSplice: accurate mapping of RNA-seq reads for splice junction discovery.

    PubMed

    Wang, Kai; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J; Huang, Yan; Savich, Gleb L; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A; Perou, Charles M; MacLeod, James N; Chiang, Derek Y; Prins, Jan F; Liu, Jinze

    2010-10-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥ 75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice. PMID:20802226

  6. Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription

    PubMed Central

    Hirochika, Hirohiko; Takatsuji, Hiroshi; Ubasawa, Aiko; Ikeda, Joh-E

    1985-01-01

    A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism. ImagesFig. 1.Fig. 3.Fig. 5.Fig. 7. PMID:16453624

  7. Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo

    SciTech Connect

    Rossi, A.M. Pisa Univ. ); Tates, A.D.; van Zeeland, A.A.; Vrieling, H. )

    1992-01-01

    Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, the authors observed (1) complete loss of one or more exons, (2) partial loss of one exon, or (3) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns.

  8. Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

    PubMed Central

    Alexandersen, S; Carpenter, S; Christensen, J; Storgaard, T; Viuff, B; Wannemuehler, Y; Belousov, J; Roth, J A

    1993-01-01

    The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown. Images PMID:8380084

  9. Alternatively Spliced Androgen Receptor Variants

    PubMed Central

    Dehm, Scott M.; Tindall, Donald J.

    2011-01-01

    Alternative splicing is an important mechanism for increasing functional diversity from a limited set of genes. De-regulation of this process is common in diverse pathologic conditions. The androgen receptor (AR) is a steroid receptor transcription factor with functions critical for normal male development as well as the growth and survival of normal and cancerous prostate tissue. Studies of AR function in androgen insensitivity syndrome (AIS) and prostate cancer (PCa) have demonstrated loss-of-function AR alterations in AIS, and gain-of-function AR alterations in PCa. Over the past two decades, AR gene alterations have been identified in various individuals with AIS, which disrupt normal AR splicing patterns and yield dysfunctional AR protein variants. More recently, altered AR splicing patterns have been identified as a mechanism of PCa progression and resistance to androgen-depletion therapy. Several studies have described the synthesis of alternatively spliced transcripts encoding truncated AR isoforms that lack the ligand-binding domain, which is the ultimate target of androgen depletion. Many of these truncated AR isoforms function as constitutively active, ligand-independent transcription factors that can support androgen-independent expression of AR target genes, as well as the androgen-independent growth of PCa cells. In this review, we will summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologic conditions of AIS and PCa. PMID:21778211

  10. Alternative splicing of RNAs transcribed from the human c- myb gene

    SciTech Connect

    Shen-Ong, G.L.C.; Skurla, R.M. Jr.; Owens, J.D.; Mushinski, J.F. )

    1990-06-01

    An alternative splicing event in which a portion of the intron bounded by the vE6 and vE7 exons with v-{ital myb} homology is included as an additional 363-nucleotide coding exon (termed E6A or coding exon 9A) has been described for normal and tumor murine cells that express {ital myb}. The authors show that this alternative splicing event is conserved in human c-{ital myb} transcripts. In addition, another novel exon (termed E7A or coding exon 10A) is identified in human c-{ital myb} mRNAs expressed in normal and tumor cells. Although the {ital myb} protein isoform encoded by murine E6A-containing mRNA is larger than the major c-{ital myb} protein, the predicted products of both forms of human alternatively spliced {ital myb} transcripts are 3{prime}-truncated {ital myb} proteins that terminate in the alternative exons. These proteins are predicted to lack the same carboxy-terminal domains as the viral {ital myb} proteins encoded by avian myeloblastosis virus and E26 virus. The junction sequences that flank these exons closely resemble the consensus splice donor and splice acceptor sequences, yet the alternative transcripts are less abundant than is the major form of c-{ital myb} transcripts. The contribution that alternative splicing events in c-{ital myb} expression may make on c-{ital myb} function remains to be elucidated.

  11. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.

    PubMed

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-09-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  12. Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

    PubMed

    Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

    2008-01-01

    Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3. PMID:18404972

  13. Donor Tag Game

    MedlinePlus

    ... Cross chapter closest to you. Can't Donate Blood? A financial donation can also help save lives. Donate Now Find ... Donation Student Donors Donation Process Eligibility Blood FAQs Blood Donor Community Learn About Blood Blood Facts and Statistics ...

  14. Splice Sites Seldom Slide: Intron Evolution in Oomycetes

    PubMed Central

    Sêton Bocco, Steven; Csűrös, Miklós

    2016-01-01

    We examine exon junctions near apparent amino acid insertions and deletions in alignments of orthologous protein-coding genes. In 1,917 ortholog families across nine oomycete genomes, 10–20% of introns are near an alignment gap, indicating at first sight that splice-site displacements are frequent. We designed a robust algorithmic procedure for the delineation of intron-containing homologous regions, and combined it with a parsimony-based reconstruction of intron loss, gain, and splice-site shift events on a phylogeny. The reconstruction implies that 12% of introns underwent an acceptor-site shift, and 10% underwent a donor-site shift. In order to offset gene annotation problems, we amended the procedure with the reannotation of intron boundaries using alignment evidence. The corresponding reconstruction involves much fewer intron gain and splice-site shift events. The frequency of acceptor- and donor-side shifts drops to 4% and 3%, respectively, which are not much different from what one would expect by random codon insertions and deletions. In other words, gaps near exon junctions are mostly artifacts of gene annotation rather than evidence of sliding intron boundaries. Our study underscores the importance of using well-supported gene structure annotations in comparative studies. When transcription evidence is not available, we propose a robust ancestral reconstruction procedure that corrects misannotated intron boundaries using sequence alignments. The results corroborate the view that boundary shifts and complete intron sliding are only accidental in eukaryotic genome evolution and have a negligible impact on protein diversity. PMID:27412607

  15. COMMUNICATION: Alternative splicing and genomic stability

    NASA Astrophysics Data System (ADS)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  16. Becoming a Donor

    MedlinePlus

    ... by Organ and Gender. > U.S. Waiting List Candidate Data HOW TO BECOME A DONOR The most important thing to do is to sign up as an organ and tissue donor in your state's donor registry. To cover all bases, it's also helpful to: Designate your decision on ...

  17. Evolution of alternative splicing after gene duplication.

    PubMed

    Su, Zhixi; Wang, Jianmin; Yu, Jun; Huang, Xiaoqiu; Gu, Xun

    2006-02-01

    Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates. PMID:16365379

  18. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  19. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  20. Traceless protein splicing utilizing evolved split inteins

    PubMed Central

    Lockless, Steve W.; Muir, Tom W.

    2009-01-01

    Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cell-based selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects. PMID:19541616

  1. The genetics of splicing in neuroblastoma

    PubMed Central

    Chen, Justin; Hackett, Christopher S.; Zhang, Shile; Song, Young K.; Bell, Robert J.A.; Molinaro, Annette M.; Quigley, David A.; Balmain, Allan; Song, Jun S.; Costello, Joseph F.; Gustafson, W. Clay; Dyke, Terry Van; Kwok, Pui-Yan; Khan, Javed; Weiss, William A.

    2015-01-01

    Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity, and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genome and exon level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from peripheral neural crest. Here we describe splicing quantitative trait loci (sQTL) associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. PMID:25637275

  2. The role of splicing factors in deregulation of alternative splicing during oncogenesis and tumor progression

    PubMed Central

    Shilo, Asaf; Siegfried, Zahava; Karni, Rotem

    2015-01-01

    In past decades, cancer research has focused on genetic alterations that are detected in malignant tissues and contribute to the initiation and progression of cancer. These changes include mutations, copy number variations, and translocations. However, it is becoming increasingly clear that epigenetic changes, including alternative splicing, play a major role in cancer development and progression. There are relatively few studies on the contribution of alternative splicing and the splicing factors that regulate this process to cancer development and progression. Recently, multiple studies have revealed altered splicing patterns in cancers and several splicing factors were found to contribute to tumor development. Studies using high-throughput genomic analysis have identified mutations in components of the core splicing machinery and in splicing factors in several cancers. In this review, we will highlight new findings on the role of alternative splicing and its regulators in cancer initiation and progression, in addition to novel approaches to correct oncogenic splicing. PMID:27308389

  3. Allelic ladder characterization of the short tandem repeat polymorphism located in the 5{prime} flanking region to the human coagulation factor XIII A subunit gene

    SciTech Connect

    Puers, C.; Lins, A.M.; Sprecher, C.J.

    1994-09-01

    The short tandem repeat (STR) polymorphism present within the 5{prime} untranslated region of the human coagulation factor XIII A subunit gene, HUM-F13A01 [AAAG]{sub n}, was evaluated using an allelic ladder, i.e., a standard size marker consisting of amplified alleles from the locus. The allelic ladder was constructed by pooling 12 polymerase chain reaction (PCR)-amplified alleles identified by their differential migration in denaturing polyacrylamide gel electrophoresis. This standard marker was used to distinguish 14 different alleles observed at this locus. Sequence analyses indicate that 13 of the alleles contain 4 through 16 iterations of the tandemly repeated AAAG sequence, respectively. The remaining allele carries four repeats and displays a deletion of two consecutive nucleotides (GT), one base distal to the repeat region. The allelic ladder was employed to type 326 F13A01 chromosomes rapidly and reliably in representatives of a German Caucasian population. Population data were analyzed with respect to Hardy-Weinberg Equilibrium (HWE) and compared with those of a previously studied Houston, Texas, Caucasian population. 27 refs., 2 figs., 1 tab.

  4. The chicken FMR1 gene is highly conserved with a CCT 5{prime} - untranslated repeat and encodes an RNA-binding protein

    SciTech Connect

    Price, D.K.; Zhang, F.; Ashley, C.T. Jr.; Warren, S.T.

    1996-01-01

    The transcriptional silencing of the human gene, fragile X metal retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5{prime}-untranslated CGG trinucleotide repeat and accounts for most cases of fragile X syndrome, a frequent inherited form of metal retardation. Although the encoded fragile X mental retardation protein (FMRP) is known to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We report the cloning of the chicken homolog of FMR1 and show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide repeat flanked on both sides by dinucleotide repeats was identified. Blocks of highly conserved 3{prime}-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both ribonucleotide particle family domains implicated in RNA binding, were identified. Chicken FMRP was found to bind RNA in vitro, and this activity correlated with the presence of the carboxy-terminal portion of the protein that includes the RGG motifs. 49 refs., 7 figs.

  5. Time-resolved spectroscopy on ultrafast proton transfer in 2-(2 prime -hydroxy-5 prime -methylphenyl)benzotriazole in liquid and polymer environments

    SciTech Connect

    Wiechmann, M.; Port, H. ); Frey, W.; Laermer, F.; Elsaesser, T. )

    1991-03-07

    The intramolecular proton transfer in 2-(2{prime}-hydroxy-5{prime}-methylphenyl)benzotriazole (TIN) is studied in different environments by stationary as well as pico- and femtosecond spectroscopy. Two ground-state conformers with a relative concentration depending on the specific solvent are distinguished from the spectra and the picosecond kinetics. The main species in nonpolar solution and in the polymer shows a weak fluorescence in the red with subpicosecond buildup and decay times. This emission is indicative of excited-state proton transfer resulting in the formation of a planar keto-type tautomer. Transient absorption measured on a femtosecond time scale gives evidence of the ultrafast keto-enol back-reaction occurring in the electronic ground state with time constants between 500 fs and 1.2 ps. In addition, a species of nonplanar geometry in the electronic ground state is detected showing emission in the blue spectral range. The fraction of the latter tautomers lies between less than 10{sup {minus}3} in nonpolar solvents and up to 60% in polar solution.

  6. Increased inosine 5{prime}-monophosphate dehydrogenase gene expression in replicating cells: A response to growth factors, not to changes in cell cycle parameters

    SciTech Connect

    Tsutani, Hiroshi; Collart, F.R.; Glesne, D.A.; Huberman, E. |

    1997-07-01

    The authors have analyzed levels of inosine 5{prime}-monophosphate dehydrogenase (IMPDH; E.C. 1.1.1.205) type II mRNA levels in a human melanoma cell line, SK-MEL-131, and a Chinese hamster ovary cell line synchronously progressing through the cell cycle following treatment with aphidicolin. Following release from the aphidicolin block at the G{sub 1}-S phase boundary, the type II IMPDH gene was found to be constitutively expressed at a similar level during all stages of the cell cycle. To analyze growth regulation, as opposed to cell cycle regulation, stable SK-MEL-131 transfectants that express a type II IMPDH-promoted heterologous construct were assayed following deprivation of serum growth factors and after restimulation with fresh serum. Serum deprivation resulted in down-regulation of both steady state type II IMPDH mRNA levels and promoter activity, while restimulation with serum resulted in up-regulation of these parameters. These findings support the conclusion that the increase in IMPDH type II gene expression in replicating cells is mainly due to growth factor regulation rather than changes in cell cycle parameters and that this regulation is mediated primarily by a transcriptional mechanism. The increased level of IMPDH expression and activity found in many tumors may therefore also be due to a transcriptionally mediated response to growth factors.

  7. In vivo footprint analysis and genomic sequencing of the human hypoxanthine-phosphoribosyl transferase (HPRT) 5 prime region on the active and inactive X chromosome

    SciTech Connect

    Hornstra, I.K.; Yang, T.P. )

    1991-03-11

    In female placental mammals, one of the two X chromosome in each somatic cell is randomly inactivated during female embryogenesis as a mechanism for dosage compensation. Once a given X chromosome is inactivated, all mitotic progeny maintain the same X chromosome in the inactive state. DNA-protein interactions and DNA methylation are hypothesized to maintain this allele-specific system of differential gene expression. Ligation-mediated polymerase chain reaction (LMPCR) in vivo footprinting and genomic sequencing were used to study DNA-protein interactions and DNA-methylation within the 5{prime} region of the X-linked human HPRT gene on the active and inactive X chromosomes. In vivo footprint analysis reveals at least one DNA-protein interaction specific to the active HPRT allele in human male fibroblast cells and hamster-human hybrid cells containing only the active human X chromosome. In the region examined, all CpG dinucleotides are methylated on the inactive HPRT allele and unmethylated on the active X allele in hamster-human hybrid cells carrying either the inactive or active human X chromosome, respectively. Thus, DNA-methylation may be mediating the differential binding of sequence-specific DNA-binding proteins to the active or inactive HPRT alleles.

  8. Donor corneal tissue evaluation.

    PubMed

    Saini, J S; Reddy, M K; Sharma, S; Wagh, S

    1996-03-01

    Proper evaluation of donor cornea is critical to the success of corneal transplantation. Attention must be paid to the cause of death and ocular condition as several general and ocular diseases constitute contraindications for donor corneal usage. Death to enucleation time should be noted. Gross examination and slit lamp biomicroscopy are mandatory for the evaluation of the donor eye while specular microscopy adds another useful dimension to information regarding donor cornea. This article provides a comprehensive review of all the aspects of donor corneal evaluation as practised today worldwide. PMID:8828299

  9. Spliced leader RNA trans-splicing discovered in copepods

    PubMed Central

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  10. Spliced leader RNA trans-splicing discovered in copepods

    NASA Astrophysics Data System (ADS)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-12-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3‧-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

  11. Spliced leader RNA trans-splicing discovered in copepods.

    PubMed

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A; Sturm, Nancy R; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3'-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  12. Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers

    SciTech Connect

    Hung, Su-Chung; Ju, Jingyue; Mathies, R.A.; Glazer, A.N.

    1996-12-01

    Energy transfer (ET) fluorescent primers are significantly superior to single dye-labeled primers for DNA sequencing and multiplex genetic analyses. We describe here ET primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes. The new ET primers have 3-({var_epsilon}-carboxy-pentyl)-3{prime}-ethyl-5,5{prime}-dimethyloxacarbocyanine (CYA; {var_epsilon}{sub M}{sup 488 nm} 142,000 M{sup -1} cm{sup -1}) at the 5{prime}-end as a common energy donor, and fluorescein or rhodamine derivatives (FAM, R6G, TAMRA, and ROX), attached to a modified thymidine 10 bases away within the primer sequence, as acceptors. With 488-nm excitation, the fluorescence emission intensity of these four ET primers is 1.4- to 24-fold stronger than that of the corresponding primers labeled only with the single acceptor dye. When compared with the corresponding ET primers with a fluorescein derivative (FAM; {var_epsilon}{sub M}{sup 488 nm} 60,000 M{sup -1} cm{sup -1}) as donor, the fluorescence emissions of primers with CYA as donor and FAM, R6G, TAMRA, and ROX as acceptors are respectively 0.8-, 1.0-, 1.7-, and 1.7-fold as intense. The low fluorescence quantum yield of the CYA donor resulted in distinct fluorescence signals for the DNA-sequencing fragments with much lower crosstalk between the four detection channels than that seen with ET primers based on a FAM donor. With single-stranded M13mp18 DNA as the template, the CYA ET primers provided DNA sequences on a four-color capillary sequencer with 100% accuracy in the first 500 bases. 15 refs., 7 figs., 1 tab.

  13. Congenital contractural arachnodactyly due to a novel splice site mutation in the FBN2 gene

    PubMed Central

    Mehar, Virendra; Yadav, Dinesh; Kumar, Ravindra; Yadav, Summi; Singh, Kuldeep; Callewaert, Bert; Pathan, Shahnawaz; De Paepe, Anne; Coucke, Paul J.

    2014-01-01

    Congenital contractural arachnodactyly is a rare autosomal dominant disorder characterized by crumpled ears, congenital contractures, arachnodactyly and scoliosis. Only few cases have been described to date. Here we report a newborn with congenital contractures, crumpled ears and scoliosis. Molecular analysis revealed a novel fibrillin-2 mutation at the donor splice site of intron 28. We discuss the differential diagnosis of neonates with congenital contractures and review the current knowledge on congenital contractural arachnodactyly.

  14. Hallmarks of alternative splicing in cancer.

    PubMed

    Oltean, S; Bates, D O

    2014-11-13

    The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors. PMID:24336324

  15. Safely splicing glass optical fibers

    NASA Technical Reports Server (NTRS)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  16. Laparoscopic live donor nephrectomy.

    PubMed

    Hasan, Waleed A; Al-Akraa, Mahmoud M

    2005-07-01

    With the number of patients presently awaiting renal transplantation exceeding the number of cadaveric organs available, there is an increasing reliance on live renal donation. Of the 11,869 renal transplants performed in 2002 in the US, 52.6% were living donors from the United Network for Organ Sharing Registry. Renal allografts from living donors provide: superior immediate long-term function; require less waiting time and are more cost-effective than those from cadaveric donors. However, anticipation of postoperative pain and temporary occupational disability may dissuade many potential donors. Additionally, some recipients hesitate to accept a living donor kidney due to suffering that would be endured by the donor. It is a unique medical situation when a young, completely healthy donor undergoes a major surgical procedure to provide an organ for transplantation. It is mandatory to offer a surgical technique, which is safe and with minimal complications. It is also obvious for any organ transplantation, that the integrity of the organ remain intact, thus, enabling its successful transplantation into the recipient. An acceptably short ischemia time and adequate lengths of ureter and renal vasculature are favored. Many centers are performing laparoscopic live donor nephrectomy in an effort to ease convalescence of renal donors. This may encourage the consideration of live donation by recipients and potential donors. PMID:16047050

  17. Lung donor selection criteria

    PubMed Central

    Chaney, John; Suzuki, Yoshikazu; Cantu, Edward

    2014-01-01

    The criteria that define acceptable physiologic and social parameters for lung donation have remained constant since their empiric determination in the 1980s. These criteria include a donor age between 25-40, a arterial partial pressure of oxygen (PaO2)/FiO2 ratio greater than 350, no smoking history, a clear chest X-ray, clean bronchoscopy, and a minimal ischemic time. Due to the paucity of organ donors, and the increasing number of patients requiring lung transplant, finding a donor that meets all of these criteria is quite rare. As such, many transplants have been performed where the donor does not meet these stringent criteria. Over the last decade, numerous reports have been published examining the effects of individual acceptance criteria on lung transplant survival and graft function. These studies suggest that there is little impact of the historical criteria on either short or long term outcomes. For age, donors should be within 18 to 64 years old. Gender may relay benefit to all female recipients especially in male to female transplants, although results are mixed in these studies. Race matched donor/recipients have improved outcomes and African American donors convey worse prognosis. Smoking donors may decrease recipient survival post transplant, but provide a life saving opportunity for recipients that may otherwise remain on the transplant waiting list. No specific gram stain or bronchoscopic findings are reflected in recipient outcomes. Chest radiographs are a poor indicator of lung donor function and should not adversely affect organ usage aside for concerns over malignancy. Ischemic time greater than six hours has no documented adverse effects on recipient mortality and should not limit donor retrieval distances. Brain dead donors and deceased donors have equivalent prognosis. Initial PaO2/FiO2 ratios less than 300 should not dissuade donor organ usage, although recruitment techniques should be implemented with intent to transplant. PMID:25132970

  18. Lung donor selection criteria.

    PubMed

    Chaney, John; Suzuki, Yoshikazu; Cantu, Edward; van Berkel, Victor

    2014-08-01

    The criteria that define acceptable physiologic and social parameters for lung donation have remained constant since their empiric determination in the 1980s. These criteria include a donor age between 25-40, a arterial partial pressure of oxygen (PaO2)/FiO2 ratio greater than 350, no smoking history, a clear chest X-ray, clean bronchoscopy, and a minimal ischemic time. Due to the paucity of organ donors, and the increasing number of patients requiring lung transplant, finding a donor that meets all of these criteria is quite rare. As such, many transplants have been performed where the donor does not meet these stringent criteria. Over the last decade, numerous reports have been published examining the effects of individual acceptance criteria on lung transplant survival and graft function. These studies suggest that there is little impact of the historical criteria on either short or long term outcomes. For age, donors should be within 18 to 64 years old. Gender may relay benefit to all female recipients especially in male to female transplants, although results are mixed in these studies. Race matched donor/recipients have improved outcomes and African American donors convey worse prognosis. Smoking donors may decrease recipient survival post transplant, but provide a life saving opportunity for recipients that may otherwise remain on the transplant waiting list. No specific gram stain or bronchoscopic findings are reflected in recipient outcomes. Chest radiographs are a poor indicator of lung donor function and should not adversely affect organ usage aside for concerns over malignancy. Ischemic time greater than six hours has no documented adverse effects on recipient mortality and should not limit donor retrieval distances. Brain dead donors and deceased donors have equivalent prognosis. Initial PaO2/FiO2 ratios less than 300 should not dissuade donor organ usage, although recruitment techniques should be implemented with intent to transplant. PMID:25132970

  19. Underwater splice for submarine coaxial cable

    SciTech Connect

    Inouye, A.T.; Roe, T. Jr.; Tausing, W.R.; Wilson, J.V.

    1984-10-30

    The invention is a device for splicing submarine coaxial cable underwater on the seafloor with a simple push-on operation to restore and maintain electrical and mechanical strength integrity; the splice device is mateable directly with the severed ends of a coaxial cable to be repaired. Splicing assemblies comprise a dielectric pressure compensating fluid filled guide cavity, a gelled castor oil cap and wiping seals for exclusion of seawater, electrical contacts, a cable strength restoration mechanism, and a pressure compensation system for controlled extrusion of and depletion loss prevention of dielectric seal fluid during cable splicing. A splice is made underwater by directly inserting prepared ends of coaxial cable, having no connector attachments, into splicing assemblies.

  20. Functional consequences of developmentally regulated alternative splicing

    PubMed Central

    Kalsotra, Auinash; Cooper, Thomas A.

    2012-01-01

    Genome-wide analyses of metazoan transcriptomes have revealed an unexpected level of mRNA diversity that is generated by alternative splicing. Recently, regulatory networks have been identified through which splicing promotes dynamic remodeling of the transcriptome to promote physiological changes, which involve robust and coordinated alternative splicing transitions. The regulation of splicing in yeast, worms, flies and vertebrates affects a variety of biological processes. The functional classes of genes that are regulated by alternative splicing include both those with widespread homeostatic activities and genes with cell-type-specific functions. Alternative splicing can drive determinative physiological change or can have a permissive role by providing mRNA variability that is utilized by other regulatory mechanisms. PMID:21921927

  1. Investigating alternative RNA splicing in Xenopus.

    PubMed

    Mereau, Agnès; Hardy, Serge

    2012-01-01

    Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors. PMID:22956098

  2. In vivo and in vitro dermal penetration of 2,4,5,2 prime ,4 prime , 5 prime -hexachlorobiphenyl in young and adult rats

    SciTech Connect

    Shah, P.V.; Sumler, M.R. ); Fisher, H.L.; Hall, L.L. )

    1989-10-01

    Penetration of 2,4,5,2{prime},4{prime},5{prime}-({sup 14}C)hexachlorobiphenyl (HCB) through skin of young (33 days) and adult (82 days) female Fischer 344 rats was determined in vivo and by two in vitro methods. In vivo dermal penetration at 120 hr was 45% in young and 43% in adults. At 72 hr in vivo dermal penetration was 35% in young and 26% in adults compared to 1.5% for young and 1.0% for adult as measured with a continuous flow in vitro system and 2.9% for young and 1.9% for adults as measured with a static in vitro system. Most of the dermally absorbed HCB remained in the body as only 4.9 and 2.6% of that absorbed was excreted by young and adult rats, respectively, at the end of 120 hr. Significant differences in dermal penetration and kinetics of HCB between young and adult female rats were observed. The elimination of ECB-derived material was approximately six times higher in feces than in urine. A physiological pharmacokinetic model was fitted to the organ and tissue radioactivity distribution data. Parameters in the model determined from dermal dosing of female Fischer 344 rats were in reasonable agreement with those reported in the literature for adult male Sprague-Dawley rats (iv dose). The rate constant for dermal penetration was 0.83 {times} 10{sup {minus}4} min{sup {minus}1} for adults and 0.96 {times} 10{sup {minus}4} min{sup {minus}1} for young. The delay or lag time parameter for dermal penetration was 4.4 hr in adults and 1.1 hr in young.

  3. Tyrosinase-positive oculocutaneous albinism in Southern African blacks: P gene-associated haplotypes suggest a major mutation in the 5{prime} region of the gene

    SciTech Connect

    Ramsay, M.; Stevens, G.; Beukering, J. van

    1994-09-01

    Tyrosinase-positive oculocutaneous albinism (ty-pos OCA) occurs with a prevalence of 1 in 3900 among Southern African (SA) blacks. The major contributors to morbidity and mortality are skin cancer and decreased visual acuity. Two distinct phenotypes occur, namely individuals with ephelides (darkly pigmented patches) and those without. There is complete concordance with regard to ephelus status among siblings. The disorder is linked to markers on chromosome 15q11.2-q12, and no obligatory cross-overs were observed with polymophic markers at the human homolog, P, of the mouse pink eyed dilute gene, p. Contrary to what has been shown for Caucasoid ty-pos OCA, this condition shows locus homogeneity among SA blacks. The P gene is an excellent candidate for ty-pos OCA and mutations in this gene will confirm its role in causing the common form of albinism in SA. Numerous P gene mutations have been described in other populations. In an attempt to detect mutations, the P gene cDNA was used to search for structural rearrangements or polymorphisms. Six polymorphisms (plR10/Scal, 912/Xbal, 912/HincII, 912/TaqI, 1412/TaqI [two systems] and 1412/HindIII) were detected with subclones of the P cDNA and haplotypes were determined in each family. None were clearly associated with an albinism-related rearrangement. However, strong linkage disequilibrium was observed with alleles at loci toward the 5{prime} region of the gene ({triangle}=0.65, 0.57 and 0.80 for the three polymorphisms detected with the 912 subclone), suggesting a major ty-pos OCA mutation in this region. Haplotype analysis provides evidence for a major mutation associated with the same haplotype in individuals with ephelides (8/12 OCA chromosomes) and those without ephelides (24:30). The presence of other ty-pos OCA associated haplotypes indicates several other less common mutations.

  4. Epistatic selection of a sequence 5{prime} of the gene responsible for cystic fibrosis may account for the high frequency of this disease in the Caucasian population

    SciTech Connect

    Macek, M. Jr. |; Nash, E.; Cutting, G.R.

    1994-09-01

    Cystic fibrosis (CF) is one of the more common lethal autosomal recessive disorders in Caucasian populations. Numerous hypotheses including genetic drift, founder effect, sex ratio, segregation distortions and various forms of heterozygote advantage have been proposed to explain the relatively high frequency of CF alleles. The observation of high linkage disequilibrium between markers at the 5{prime} end of CFTR and mutations that cause CF raised the possibility of epistatic selection. CF-linked marker allele frequencies were determined in 417 elderly individuals from a stable Czech population that survived high levels of infant and childhood mortality in the pre-antibiotic era. These data were compared with allele frequencies of 646 contemporary newborns and 345 young adults drawn from the same population who had significantly lower mortality rates in the antibiotic era. Allele frequencies of markers CS7/Hhal and KM19/Pstl from the D7S23 locus are significantly different (p<0.05) between elderly female and male subjects in this population. Furthermore, there is a significant difference in the allele frequencies of marker CS7/Hhal when newborn females and elderly women are compared (p<0.05). Taken together, these data suggest that the allele status at the CS7 region influenced female survival in the period of high infant and childhood mortality in the pre-antibiotic era. Under this selective pressure, CFTR mutations that occurred on the {open_quotes}favorable{close_quotes} background would marginally increase in frequency in each successive generation and more ancient mutations residing on this background would become the most frequent in the general population.

  5. The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

    SciTech Connect

    Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.

    2009-09-02

    The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N{sub x})AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5{prime} splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine 'ladder.' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

  6. Molecular aspects of DNA splicing system

    NASA Astrophysics Data System (ADS)

    Yusof, Yuhani; Lim, Wen Li; Goode, T. Elizabeth; Sarmin, Nor Haniza; Heng, Fong Wan; Wahab, Mohd Firdaus Abd

    2015-05-01

    The pioneer model of deoxyribonucleic acid (DNA) splicing system in a framework of Formal Language Theory was introduced by Head that led to the existence of other models of splicing system, namely Paun, Pixton and Yusof-Goode. These entire models are inspired by the molecular biological process of DNA splicing. Hence, this paper focuses on the translucent DNA splicing process, particularly on the generated language. Starting with some preliminaries in a limit graph, this paper also provides the experimental design with the predicted and actual result.

  7. Recursive splicing in long vertebrate genes

    PubMed Central

    Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-01-01

    It is generally believed that splicing removes introns as single units from pre-mRNA transcripts. However, some long D. melanogaster introns contain a cryptic site, called a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing1,2. The extent to which recursive splicing occurs in other species and its mechanistic basis remain unclear. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of a “RS-exon” that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform due to competition with a reconstituted 5′ splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic exons or promoters that are prevalent in long introns, but which fail to reconstitute an efficient 5′ splice site. Most RS-exons contain a premature stop codon such that their inclusion may decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling inclusion of cryptic elements with RS-exons. PMID:25970246

  8. Tropomyosin exons as models for alternative splicing.

    PubMed

    Gooding, Clare; Smith, Christopher W J

    2008-01-01

    Three of the four mammalian tropomyosin (Tm) genes are alternatively spliced, most commonly by mutually exclusive selection from pairs of internal or 3' end exons. Alternative splicing events in the TPM1, 2 and 3 genes have been analysed experimentally in various levels ofdetail. In particular, mutually exclusive exon pairs in the betaTm (TPM2) and alphaTm (TPM1) genes are among the most intensively studied models for striated and smooth muscle specific alternative splicing, respectively. Analysis of these model systems has provided important insights into general mechanisms and strategies of splicing regulation. PMID:19209811

  9. Recursive splicing in long vertebrate genes.

    PubMed

    Sibley, Christopher R; Emmett, Warren; Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons. PMID:25970246

  10. The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing.

    PubMed Central

    Hodges, P E; Rosenberg, L E

    1989-01-01

    Ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is a mitochondrial matrix enzyme of the mammalian urea cycle. The X chromosome-linked spfash mutation in the mouse causes partial ornithine transcarbamylase deficiency and has served as a model for the human disease. We show here that the spfash mutation is a guanine to adenine transition of the last nucleotide of the fourth exon of the ornithine transcarbamylase gene. This nucleotide change produces two remarkably different effects. First, this transition causes ornithine transcarbamylase mRNA deficiency because the involved exon nucleotide plays a part in the recognition of the adjacent splice donor site. As a result of the mutation, ornithine transcarbamylase pre-mRNA is spliced inefficiently both at this site and at a cryptic splice donor site 48 bases into the adjacent intron. Second, two mutant proteins are translated from these mRNAs. From the correctly spliced mRNA, the transition results in a change of amino acid 129 from arginine to histidine. This missense substitution has no discernable effect on mitochondrial import, subunit assembly, or enzyme activity. On the other hand, the elongated mRNA resulting from mis-splicing is translated into a dysfunctional ornithine transcarbamylase subunit elongated by the insertion of 16 amino acid residues. Images PMID:2471197

  11. Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible

    PubMed Central

    Lareau, Liana F.; Brenner, Steven E.

    2015-01-01

    Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. PMID:25576366

  12. Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.

    PubMed

    Rimoldi, Valeria; Straniero, Letizia; Asselta, Rosanna; Mauri, Lucia; Manfredini, Emanuela; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Soldà, Giulia; Primignani, Paola

    2014-03-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants. PMID:24361966

  13. The splice is right: Guarantors of fidelity in pre-mRNA splicing

    PubMed Central

    Horowitz, David S.

    2011-01-01

    Two recent papers, one from the Staley laboratory (Koodathingal and colleagues) and the other from the Cheng laboratory (Tseng and colleagues), show that the RNA-dependent ATPase Prp16, which is required for the second step of splicing, acts to reject slowly splicing pre-mRNAs immediately before the first catalytic reaction in pre-mRNA splicing. The results answer long-investigated questions about the actions of Prp16 and provide a wealth of molecular details on the proofreading process in pre-mRNA splicing. The discussion here reviews and integrates the results of the two papers and describes the implications for proofreading in splicing. PMID:21357751

  14. Rich Donors, Poor Countries

    ERIC Educational Resources Information Center

    Thomas, M. A.

    2012-01-01

    The shifting ideological winds of foreign aid donors have driven their policy towards governments in poor countries. Donors supported state-led development policies in poor countries from the 1940s to the 1970s; market and private-sector driven reforms during the 1980s and 1990s; and returned their attention to the state with an emphasis on…

  15. Donor Telomere Length SAA

    Cancer.gov

    A new NCI study has found that, among patients with severe aplastic anemia who received a hematopoietic cell transplant from an unrelated donor, those whose donor white blood cells had longer telomeres had higher survival rates five-years after transplant

  16. Donor selection and management.

    PubMed

    Snell, Gregory I; Paraskeva, Miranda; Westall, Glen P

    2013-06-01

    This article reviews recent developments in the selection, assessment, and management of the potential lung donor, which aim to increase donor organ use. The scarcity of suitable donor organs continues to limit lung transplantation, but the situation is changing. An expanded donor pool, including the now widespread use of donation after cardiac death (DCD) lungs; the use of extended donor lungs; and the ability of ex vivo lung perfusion (EVLP) to evaluate and improve donor lungs are key initiatives. These strategies have substantially lifted donor lung utilization rates from historically low levels of less than 15% to rates greater than 50%. Indeed, since 2004 there has been an accelerated year-on-year increase in the number of lungs transplanted globally. Intermediate-term studies are now confirming that long-term outcomes are not being significantly compromised and that more individuals with terminal, symptomatic lung disease are being transplanted. It is now quite clear that many of the historical factors used to define a lung as "extended" do not actually produce significantly inferior outcomes. There has been a dramatic increase in research and clinical interest in donor lung assessment, management, and novel therapeutic strategies. The lessons learned are now being applied widely beyond the lung as researchers aim to increase availability and optimize other solid organs for transplantation. PMID:23821510

  17. Cutting, Splicing, and Kelvin Waves

    NASA Astrophysics Data System (ADS)

    Scheeler, Martin; Kleckner, Dustin; Irvine, William T. M.

    2013-11-01

    Recent experimental advances have allowed us to create, visualize and track vortices of prescribed shape and topology in classical fluids. We study the effect of surgery (cutting and splicing) on the evolution of the geometry and topology of these vortex loops, with a particular focus on the wave-like excitations generated by these operations. We interpret the dynamics of these excitations and the role they play within the broader context of vortex evolution. This work was supported by the National Science Foundation Materials Research and Engineering Centers (MRSEC) Program at the University of Chicago (DMR-0820054) and the Packard Foundation through a Packard fellowship.

  18. HS3D, A Dataset of Homo Sapiens Splice Regions, and its Extraction Procedure from a Major Public Database

    NASA Astrophysics Data System (ADS)

    Pollastro, Pasquale; Rampone, Salvatore

    The aim of this work is to describe a cleaning procedure of GenBank data, producing material to train and to assess the prediction accuracy of computational approaches for gene characterization. A procedure (GenBank2HS3D) has been defined, producing a dataset (HS3D - Homo Sapiens Splice Sites Dataset) of Homo Sapiens Splice regions extracted from GenBank (Rel.123 at this time). It selects, from the complete GenBank Primate Division, entries of Human Nuclear DNA according with several assessed criteria; then it extracts exons and introns from these entries (actually 4523 + 3802). Donor and acceptor sites are then extracted as windows of 140 nucleotides around each splice site (3799 + 3799). After discarding windows not including canonical GT-AG junctions (65 + 74), including insufficient data (not enough material for a 140 nucleotide window) (686 + 589), including not AGCT bases (29 + 30), and redundant (218 + 226), the remaining windows (2796 + 2880) are reported in the dataset. Finally, windows of false splice sites are selected by searching canonical GT-AG pairs in not splicing positions (271 937 + 332 296). The false sites in a range +/- 60 from a true splice site are marked as proximal. HS3D, release 1.2 at this time, is available at the Web server of the University of Sannio: http://www.sci.unisannio.it/docenti/rampone/.

  19. The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans.

    PubMed

    Tchilian, E Z; Wallace, D L; Imami, N; Liao, H X; Burton, C; Gotch, F; Martinson, J; Haynes, B F; Beverley, P C

    2001-05-15

    The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted. PMID:11342634

  20. SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

    PubMed Central

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-01-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

  1. Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

    PubMed Central

    Comiskey, Daniel F.; Jacob, Aishwarya G.; Singh, Ravi K.; Tapia-Santos, Aixa S.; Chandler, Dawn S.

    2015-01-01

    Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590

  2. Functional selection of splicing enhancers that stimulate trans-splicing in vitro.

    PubMed Central

    Boukis, L A; Bruzik, J P

    2001-01-01

    The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element (G/C)GAC(G/C) and also 5' splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5' end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5' splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction. PMID:11421358

  3. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    PubMed

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations. PMID:26300000

  4. Subgroup Specific Alternative Splicing in Medulloblastoma

    PubMed Central

    Kloosterhof, Nanne K; Northcott, Paul A; Yu, Emily PY; Shih, David; Peacock, John; Grajkowska, Wieslawa; van Meter, Timothy; Eberhart, Charles G; Pfister, Stefan; Marra, Marco A; Weiss, William A; Scherer, Stephen W; Rutka, James T; French, Pim J; Taylor, Michael D

    2014-01-01

    Medulloblastoma is comprised of four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P<6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals over-representation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups. PMID:22358458

  5. Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets

    PubMed Central

    Gillies, Christopher; Sampson, Matthew G.; Kher, Vijay; Sethi, Sidharth K.; Otto, Edgar A.

    2015-01-01

    Objective Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. Methods We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. Results WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient’s EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human

  6. Phosphoregulation of Ire1 RNase splicing activity

    NASA Astrophysics Data System (ADS)

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-04-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation.

  7. The Characterizations of Different Splicing Systems

    NASA Astrophysics Data System (ADS)

    Karimi, Fariba; Sarmin, Nor Haniza; Heng, Fong Wan

    The concept of splicing system was first introduced by Head in 1987 to model the biological process of DNA recombination mathematically. This model was made on the basis of formal language theory which is a branch of applied discrete mathematics and theoretical computer science. In fact, splicing system treats DNA molecule and the recombinant behavior by restriction enzymes and ligases in the form of words and splicing rules respectively. The notion of splicing systems was taken into account from different points of view by many mathematicians. Several modified definitions have been introduced by many researchers. In this paper, some properties of different kinds of splicing systems are presented and their relationships are investigated. Furthermore, these results are illustrated by some examples.

  8. Structural analysis of covalent peptide dimers, bis(pyridine-2-carboxamidonetropsin)(CH[sub 2])[sub 3][sup 6], in complex with 5[prime]-TGACT-3[prime] sites by two-dimensional NMR

    SciTech Connect

    Dwyer, T.J.; Geierstanger, B.H.; Wemmer, D.E. ); Mrksich, M.; Dervan, P.B. )

    1993-11-03

    The peptide pyridine-2-carboxamidonetropsin (2-PyN) binds specifically in the minor groove of 5[prime]-(A,T)G-(A,T)C(A,T)-3[prime] sequences as a side-by-side antiparallel dimer. Tethering two 2-PyN ligands through the nitrogens of the central pyrrole rings with propyl, butyl, pentyl and hexyl linkers affords covalent peptide dimers, bis(pyridine-2-carboxamide-netropsin)(CH[sub 2])[sub 3[minus]6], which bind in the minor groove of DNA with increased binding affinities and improved sequence specificities. Two-dimensional NMR studies of the complexes formed upon binding of these covalent peptide dimers to an oligonucleotide containing a 5[prime]-TGACT-3[prime] site reveal that the dimeric peptides bind as intramolecular dimers with nearly identical geometry and peptide-DNA contacts as in the (2-PyN)[sub 2][center dot]5[prime]-TGACT-3[prime] complex. 13 refs., 9 figs., 2 tabs.

  9. Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

    PubMed Central

    Wang, Zefeng; Burge, Christopher B.

    2008-01-01

    Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or “code” for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions. PMID:18369186

  10. Role of the 3′ Splice Site in U12-Dependent Intron Splicing

    PubMed Central

    Dietrich, Rosemary C.; Peris, Marian J.; Seyboldt, Andrew S.; Padgett, Richard A.

    2001-01-01

    U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site. PMID:11238930

  11. Living Donor Liver Transplantation

    MedlinePlus

    ... around the scar. The bulges can usually be fixed with surgery. During your medical exam, ask the ... to find out if the donor's blood type matches the recipient’s blood type. Next, the transplant team ...

  12. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts. PMID:16951290

  13. CYP17A1 intron mutation causing cryptic splicing in 17α-hydroxylase deficiency.

    PubMed

    Hwang, Daw-Yang; Hung, Chi-Chih; Riepe, Felix G; Auchus, Richard J; Kulle, Alexandra E; Holterhus, Paul-Martin; Chao, Mei-Chyn; Kuo, Mei-Chuan; Hwang, Shang-Jyh; Chen, Hung-Chun

    2011-01-01

    17α-Hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination. PMID:21966534

  14. CYP17A1 Intron Mutation Causing Cryptic Splicing in 17α-Hydroxylase Deficiency

    PubMed Central

    Hwang, Daw-Yang; Hung, Chi-Chih; Riepe, Felix G.; Auchus, Richard J.; Kulle, Alexandra E.; Holterhus, Paul-Martin; Chao, Mei-Chyn; Kuo, Mei-Chuan; Hwang, Shang-Jyh; Chen, Hung-Chun

    2011-01-01

    17α-hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination. PMID:21966534

  15. Fibronectin Splice Variation in Human Knee Cartilage, Meniscus and Synovial Membrane: Observations in Osteoarthritic Knee

    PubMed Central

    Scanzello, Carla R.; Markova, Dessislava Z.; Chee, Ana; Xiu, Yan; Adams, Sherrill L.; Anderson, Greg; Zgonis, Miltiadis; Qin, Ling; An, Howard S.; Zhang, Yejia

    2014-01-01

    Objective Fibronectin (FN) is a widely expressed molecule that can participate in development of osteoarthritis (OA) affecting cartilage, meniscus, and synovial membrane (SM). The alternatively spliced isoforms of FN in joint tissues other than cartilage have not been extensively studied previously. The present study compares FN splice variation in patients with varying degrees of osteoarthritic change. Design Joint tissues were collected from asymptomatic donors and patients undergoing arthroscopic procedures. Total RNA was amplified by PCR using primers flanking alternatively spliced Extra Domain A (EDA), Extra Domain B (EDB) and Variable (V) regions. Results EDB+, EDB− and EDA− and V+ variants were present in all joint tissues, while the EDA+ variant was rarely detected. Expression levels of EDB+ and EDV+ variants were similar in cartilage, synovium and meniscal tissues. Synovial expression of V+ FN in arthroscopy patients varied with degree of cartilage degeneration. Two V− isoforms, previously identified in cartilage, were also present in SM and meniscus. Conclusions Fibronectin splicing in meniscus and SM bears striking resemblance to that of cartilage. Expression levels of synovial V+ FN varied with degree of cartilage degeneration. V+ FN should be investigated as a potential biomarker of disease stage or progression in larger populations. PMID:25410897

  16. Splicing of Retrotransposon Insertions from Transcripts of the Drosophila Melanogaster Vermilion Gene in a Revertant

    PubMed Central

    Pret, A. M.; Searles, L. L.

    1991-01-01

    A mutation of the Drosophila melanogaster vermilion (v) gene known as v(1) is caused by the insertion of a 412 retrotransposon into the 5' untranslated region of the first exon. Mutants carrying this insertion accumulate a low level of mRNA from which most of the transposon sequences have been eliminated by splicing at cryptic sites within transposon sequences. Here, we demonstrate that a revertant of the v(1) allele called v(+37) is caused by the insertion of a second retrotransposon, the B104/roo element, into a site near one end of the 412 element. The revertant strain accumulates a higher level of mRNA from which most of both transposons have been removed by splicing at new donor sites introduced by the B104/roo insertion and the same acceptor site within 412. Mutations at suppressor of sable [su(s)], which increase the accumulation of v(1) transcripts, slightly elevate the level of v(+37) RNA. In addition, we show that the first v intron downstream of the 412 insertion is not efficiently removed in the v(1) mutant, and suppressor and reversion mutations increase the proportion of transcripts that are properly spliced at that downstream intron. Thus, it appears that both the suppressor and reversion mutations exert an effect at the level of pre-mRNA splicing. PMID:1664404

  17. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  18. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  19. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  20. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  1. Laparoscopic donor nephrectomy.

    PubMed

    Deger, S; Giessing, M; Roigas, J; Wille, A H; Lein, M; Schönberger, B; Loening, S A

    2005-01-01

    Laparoscopic live donor nephrectomy (LDN) has removed disincentives of potential donors and may bear the potential to increase kidney donation. Multiple modifications have been made to abbreviate the learning curve while at the same time guarantee the highest possible level of medical quality for donor and recipient. We reviewed the literature for the evolution of the different LDN techniques and their impact on donor, graft and operating surgeon, including the subtleties of different surgical accesses, vessel handling and organ extraction. We performed a literature search (PubMed, DIMDI, medline) to evaluate the development of the LDN techniques from 1995 to 2003. Today more than 200 centres worldwide perform LDN. Hand-assistance has led to a spread of LDN. Studies comparing open and hand-assisted LDN show a reduction of operating and warm ischaemia times for the hand-assisted LDN. Different surgical access sites (trans- or retroperitoneal), different vessel dissection approaches, donor organ delivery techniques, delivery sites and variations of hand-assistance techniques reflect the evolution of LDN. Proper techniques and their combination for the consecutive surgical steps minimize both warm ischaemia time and operating time while offering the donor a safe minimally invasive laparoscopic procedure. LDN has breathed new life into the moribund field of living kidney donation. Within a few years LDN could become the standard approach in living kidney donation. Surgeons working in this field must be trained thoroughly and well acquainted with the subtleties of the different LDN techniques and their respective advantages and disadvantages. PMID:16754618

  2. Live-donor nephrectomy.

    PubMed

    Rocca, Juan P; Davis, Eric; Edye, Michael

    2012-01-01

    Six decades after its first implementation, kidney transplantation remains the optimal therapy for end-stage renal disease requiring dialysis. Despite the incontrovertible mortality reduction and cost-effectiveness of kidney transplantation, the greatest remaining barrier to treatment of end-stage renal disease is organ availability. Although the waiting list of patients who stand to benefit from kidney transplantation grows at a rate proportional to the overall population and proliferation of diabetes and hypertension, the pool of deceased-donor organs available for transplantation experiences minimal to no growth. Because the kidney is uniquely suited as a paired organ, the transplant community's answer to this shortage is living donation of a healthy volunteer's kidney to a recipient with end-stage renal disease. This review details the history and evolution of living-donor kidney transplantation in the United States as well as advances the next decade promises. Laparoscopic donor nephrectomy has overcome many of the obstacles to living donation in terms of donor morbidity and volunteerism. Known donor risks in terms of surgical and medical morbidity are reviewed, as well as the ongoing efforts to delineate and mitigate donor risk in the context of accumulating recipient morbidity while on the waiting list. PMID:22678857

  3. Aberrant splicing and drug resistance in AML.

    PubMed

    de Necochea-Campion, Rosalia; Shouse, Geoffrey P; Zhou, Qi; Mirshahidi, Saied; Chen, Chien-Shing

    2016-01-01

    The advent of next-generation sequencing technologies has unveiled a new window into the heterogeneity of acute myeloid leukemia (AML). In particular, recurrent mutations in spliceosome machinery and genome-wide aberrant splicing events have been recognized as a prominent component of this disease. This review will focus on how these factors influence drug resistance through altered splicing of tumor suppressor and oncogenes and dysregulation of the apoptotic signaling network. A better understanding of these factors in disease progression is necessary to design appropriate therapeutic strategies recognizing specific alternatively spliced or mutated oncogenic targets. PMID:27613060

  4. Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

    NASA Astrophysics Data System (ADS)

    Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

    1994-09-01

    The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

  5. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

    Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.

    2015-01-01

    Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083

  6. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

    PubMed Central

    Humphrey, M B; Bryan, J; Cooper, T A; Berget, S M

    1995-01-01

    Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon. PMID:7623794

  7. Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures

    SciTech Connect

    Hunter, T.C.; Albertini, R.J.; O`Neill, J.P.

    1996-03-01

    Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D. 16 refs., 3 tabs.

  8. Distinctive Characteristics of Educational Donors

    ERIC Educational Resources Information Center

    James, Russell N., III.

    2008-01-01

    Examining the charitable behavior of 56,663 US households, this paper evaluates the distinctive characteristics of educational donors as compared with donors to noneducational charitable organizations and with nondonors. In general, educational donors had significantly greater income, wealth, and education than other donors. Educational donors…

  9. Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing.

    PubMed

    Munding, Elizabeth M; Shiue, Lily; Katzman, Sol; Donohue, John Paul; Ares, Manuel

    2013-08-01

    During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing. PMID:23891561

  10. Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants.

    PubMed

    Vallée, Maxime P; Di Sera, Tonya L; Nix, David A; Paquette, Andrew M; Parsons, Michael T; Bell, Russel; Hoffman, Andrea; Hogervorst, Frans B L; Goldgar, David E; Spurdle, Amanda B; Tavtigian, Sean V

    2016-07-01

    Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs-the "integrated evaluation"-that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at http://hci-exlovd.hci.utah.edu, with 77 re-evaluable variants. PMID:26913838

  11. Characterization of a novel TYMP splice site mutation associated with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

    PubMed

    Taanman, Jan-Willem; Daras, Mariza; Albrecht, Juliane; Davie, Charles A; Mallam, Elizabeth A; Muddle, John R; Weatherall, Mark; Warner, Thomas T; Schapira, Anthony H V; Ginsberg, Lionel

    2009-02-01

    Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive disorder caused by loss-of-function mutations in the thymidine phosphorylase gene (TYMP). We report here a patient compound heterozygous for two TYMP mutations: a novel g.4009G>A transition affecting the consensus splice donor site of intron 9, and a previously reported g.675G>C splice site mutation. The novel mutation causes exon 9 skipping but leaves the reading frame intact; however, TYMP protein was not detected by immunoblot analysis, suggesting that neither mutant allele is expressed as protein. The patient's fibroblasts showed gradual loss of the mitochondrial DNA-encoded subunit I of cytochrome-c oxidase, suggesting a progressive mitochondrial DNA defect in culture. PMID:19056268

  12. Molecular characterization of novel splice site mutation causing protein C deficiency.

    PubMed

    Al-Hamed, Mohamed H; AlBatniji, Fatma; AlDakheel, Ghadah A; El-Faraidi, Huda; Al-Zahrani, Azzah; Al-Abbass, Fahed; Imtiaz, Faiqa

    2016-07-01

    Congenital protein C deficiency is an inherited coagulation disorder associated with an elevated risk of venous thromboembolism. A Saudi Arabian male from a consanguineous family was admitted to neonatal intensive care unit in his first days of life because of transient tachypnea and hematuria. Laboratory investigations determined low platelet and protein C deficiency. Direct sequencing of PROC gene and RNA analysis were performed. Analysis of factor V Leiden (G1691A) and factor II (G20210A) mutations was also done. Novel homozygous splice site mutation c.796+3A>T was detected in the index case and segregation was confirmed in the family. RNA analysis revealed the pathogenicity of the mutation by skipping exon 8 of PROC gene and changing the donor splice site of the exon. Detection of the molecular cause of protein C deficiency reduces life threatening and facilitates inductive carrier testing, prenatal and preimplantation genetic diagnosis for families. PMID:26656900

  13. Group II Intron Self-Splicing.

    PubMed

    Pyle, Anna Marie

    2016-07-01

    Group II introns are large, autocatalytic ribozymes that catalyze RNA splicing and retrotransposition. Splicing by group II introns plays a major role in the metabolism of plants, fungi, and yeast and contributes to genetic variation in many bacteria. Group II introns have played a major role in genome evolution, as they are likely progenitors of spliceosomal introns, retroelements, and other machinery that controls genetic variation and stability. The structure and catalytic mechanism of group II introns have recently been elucidated through a combination of genetics, chemical biology, solution biochemistry, and crystallography. These studies reveal a dynamic machine that cycles progressively through multiple conformations as it stimulates the various stages of splicing. A central active site, containing a reactive metal ion cluster, catalyzes both steps of self-splicing. These studies provide insights into RNA structure, folding, and catalysis, as they raise new questions about the behavior of RNA machines. PMID:27391926

  14. Lessons from non-canonical splicing.

    PubMed

    Sibley, Christopher R; Blazquez, Lorea; Ule, Jernej

    2016-07-01

    Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies. PMID:27240813

  15. Molecular analysis of human argininosuccinate lyase: Mutant characterization and alternative splicing of the coding region

    SciTech Connect

    Walker, D.C. ); McCloskey, D.A.; Simard, L.R.; McInnes, R.R. )

    1990-12-01

    Argininosuccinic acid lyase (ASAL) deficiency is a clinically heterogeneous autosomal recessive urea cycle disorder. The authors previously established by complementation analysis that 29 ASAL-deficient patients have heterogeneous mutations in a single gene. To prove that the ASAL structural gene is the affected locus, they sequenced polymerase chain reaction-amplified ASAL cDNA of a representative mutant from the single complementation group. Fibroblast strain 944 from a late-onset patient who was the product of a consanguineous mating, had only a single base-pair change in the coding region, a C-283{r arrow} T transition at a CpG dinucleotide in exon 3. This substitution converts Arg-95 to Cys (R95C), occurs in a stretch of 13 residues that is identical in yeast and human ASAL, and was present in both of the patient's alleles but not in 14 other mutant or 10 normal alleles. They observed that amplified cDNA from mutant 944 and normal cells (liver, keratinocytes, lymphoblasts, and fibroblasts) contained, in addition to the expected 5{prime} 513-base-pair band, a prominent 318-base-pair ASAL band formed by the splicing of exon 2 from the transcript. The short transcript maintains the ASAL reading frame but removes Lys-51, a residue that may be essential for catalysis, since it binds the argininosuccinate substrate. They conclude (i) that the identification of the R95C mutation in strain 944 demonstrates that virtually all ASAL deficiency results from defects in the ASAL structural gene and (ii) that minor alternative splicing of the coding region occurs at the ASAL locus.

  16. Systems of donor transfer.

    PubMed

    de Charro, F T; Akveld, H E; Hessing, D J

    1993-10-01

    The development of medical knowledge has resulted in a demand in society for donor organs, but the recruitment of donor organs for transplantation is difficult. This paper aims to provide some general insights into the complex interaction processes involved. A laissez-faire policy, in which market forces are relied on, is not acceptable from an ethical and legal point of view in most western European countries. Especially at the demand side of the exchange of donor organs, commercialism is to be opposed. We judge the use of commercial incentives at the supply side less unacceptable in theory but not feasible in western European countries. Since market forces are deemed unacceptable as instruments for coordinating demand and supply of donor organs, donor procurement has to be considered as a collective good, and therefore governments are faced with the responsibility of making sure that alternative interaction and distribution mechanisms function. The role of organ procurement agencies (OPAs) in societal interaction concerning postmortem organ donation is described using a two-dimensional conceptualisation scheme. Medical aspects of living organ donation are described. An international comparative description of legal systems to regulate living organ donation in western European countries completes this survey. PMID:10129766

  17. Independent donor ethical assessment: aiming to standardize donor advocacy.

    PubMed

    Choudhury, Devasmita; Jotterand, Fabrice; Casenave, Gerald; Smith-Morris, Carolyn

    2014-06-01

    Living organ donation has become more common across the world. To ensure an informed consent process, given the complex issues involved with organ donation, independent donor advocacy is required. The choice of how donor advocacy is administered is left up to each transplant center. This article presents the experience and process of donor advocacy at University of Texas Southwestern Medical Center administered by a multidisciplinary team consisting of physicians, surgeons, psychologists, medical ethicists and anthropologists, lawyers, a chaplain, a living kidney donor, and a kidney transplant recipient. To ensure that advocacy remains fair and consistent for all donors being considered, the donor advocacy team at University of Texas Southwestern Medical Center developed the Independent Donor Ethical Assessment, a tool that may be useful to others in rendering donor advocacy. In addition, the tool may be modified as circumstances arise to improve donor advocacy and maintain uniformity in decision making. PMID:24919733

  18. Managing finances of shipping living donor kidneys for donor exchanges.

    PubMed

    Mast, D A; Vaughan, W; Busque, S; Veale, J L; Roberts, J P; Straube, B M; Flores, N; Canari, C; Levy, E; Tietjen, A; Hil, G; Melcher, M L

    2011-09-01

    Kidney donor exchanges enable recipients with immunologically incompatible donors to receive compatible living donor grafts; however, the financial management of these exchanges, especially when an organ is shipped, is complex and thus has the potential to impede the broader implementation of donor exchange programs. Representatives from transplant centers that utilize the National Kidney Registry database to facilitate donor exchange transplants developed a financial model applicable to paired donor exchanges and donor chain transplants. The first tenet of the model is to eliminate financial liability to the donor. Thereafter, it accounts for the donor evaluation, donor nephrectomy hospital costs, donor nephrectomy physician fees, organ transport, donor complications and recipient inpatient services. Billing between hospitals is based on Medicare cost report defined costs rather than charges. We believe that this model complies with current federal regulations and effectively captures costs of the donor and recipient services. It could be considered as a financial paradigm for the United Network for Organ Sharing managed donor exchange program. PMID:21831153

  19. Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

    PubMed Central

    Kameyama, Toshiki; Suzuki, Hitoshi; Mayeda, Akila

    2012-01-01

    Transcripts of the human tumor susceptibility gene 101 (TSG101) are aberrantly spliced in many cancers. A major aberrant splicing event on the TSG101 pre-mRNA involves joining of distant alternative 5′ and 3′ splice sites within exon 2 and exon 9, respectively, resulting in the extensive elimination of the mRNA. The estimated strengths of the alternative splice sites are much lower than those of authentic splice sites. We observed that the equivalent aberrant mRNA could be generated from an intron-less TSG101 gene expressed ectopically in breast cancer cells. Remarkably, we identified a pathway-specific endogenous lariat RNA consisting solely of exonic sequences, predicted to be generated by a re-splicing between exon 2 and exon 9 on the spliced mRNA. Our results provide evidence for a two-step splicing pathway in which the initial constitutive splicing removes all 14 authentic splice sites, thereby bringing the weak alternative splice sites into close proximity. We also demonstrate that aberrant multiple-exon skipping of the fragile histidine triad (FHIT) pre-mRNA in cancer cells occurs via re-splicing of spliced FHIT mRNA. The re-splicing of mature mRNA can potentially generate mutation-independent diversity in cancer transcriptomes. Conversely, a mechanism may exist in normal cells to prevent potentially deleterious mRNA re-splicing events. PMID:22675076

  20. Commitment of apolipoprotein B RNA to the splicing pathway regulates cytidine-to-uridine editing-site utilization.

    PubMed Central

    Sowden, M P; Smith, H C

    2001-01-01

    A tripartite motif located in the centre of the 7.5 kb exon 26 of apolipoprotein B (apoB) mRNA directs editosome assembly and site-specific cytidine-to-uridine editing at nucleotide 6666. apoB mRNA editing is a post-transcriptional event, occurring primarily at the time exon 26 is spliced or at a time after splicing, but before nuclear export. We show, through reporter RNA constructs, that RNA splice sites suppress editing of precursor RNAs when placed proximal or distal to the editing site. Processed RNAs were edited more efficiently than precursor RNAs. Mutation of both the splice donor and acceptor sites was necessary for RNAs to be edited efficiently. The results suggested that commitment of pre-mRNA to the splicing and/or nuclear-export pathways may play a role in regulating editing-site utilization. The HIV-1 Rev-Rev response element ('RRE') interaction was utilized to uncouple the commitment of precursor RNAs to the spliceosome assembly pathway and associated nuclear-export pathway. Under these conditions, unspliced reporter RNAs were edited efficiently. We propose that pre-mRNA passage through the temporal or spatial restriction point where they become committed to spliceosome assembly contributes regulatory information for subsequent editosome activity. PMID:11672445

  1. Alternative splicing of Wilms tumor suppressor 1 (Wt1) exon 4 results in protein isoforms with different functions.

    PubMed

    Schnerwitzki, Danny; Perner, Birgit; Hoppe, Beate; Pietsch, Stefan; Mehringer, Rebecca; Hänel, Frank; Englert, Christoph

    2014-09-01

    The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor that is essential for development of multiple organs including kidneys, gonads, spleen and heart. In mammals Wt1 comprises 10 exons with two characteristic splicing events: inclusion or skipping of exon 5 and alternative usage of two splice donor sites between exons 9 and 10. Most fish including zebrafish and medaka possess two wt1 paralogs, wt1a and wt1b, both lacking exon 5. Here we have characterized wt1 in guppy, platyfish and the short-lived African killifish Nothobranchius furzeri. All fish except zebrafish show alternative splicing of exon 4 of wt1a but not of wt1b with the wt1a(-exon 4) isoform being the predominant splice variant. With regard to function, Wt1a(+exon 4) showed less dimerization but stimulated transcription more effectively than the Wt1a(-exon 4) isoform. A specific knockdown of wt1a exon 4 in zebrafish was associated with anomalies in kidney development demonstrating a physiological function for Wt1a exon 4. Interestingly, alternative splicing of exon 4 seems to be an early evolutionary event as it is observed in the single wt1 gene of the sturgeon, a species that has not gone through teleost-specific genome duplication. PMID:25014653

  2. HMG-CoA lyase (HL) gene: Cloning and characterization of the 5{prime} end of the mouse gene, gene targeting in ES cells, and demonstration of large deletions in three HL-deficient patients

    SciTech Connect

    Wang, S.; Robert, M.F.; Mitchell, G.A.

    1994-09-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL) is a mitochondrial matrix enzyme which catalyzes the last step of leucine catabolism and of ketogenesis. Autosomal recessive HL deficiency in humans results in episodes of hypoglycemia and coma. We are interested in the pathophysiology of HL deficiency as a model for both amino acid and fatty acid inborn errors. We have cloned the human and mouse HL genes. In order to analyze the 5{prime} nontranslated region of mouse HL gene, we cloned and sequenced a 1.8 kb fragment containing the 5{prime} extremity including exon 1 and about 1.6 kb of 5{prime} nontranslated sequence. The region surrounding exon 1 is CpG-rich (66.4%). Using the criteria of West, the Observed/Expected ratio for CpG dinucleotides is 0.7 ({ge}0.6 is consistent with a CpG island). We are carrying out primer extension and RNase protection experiments to determine the transcription initiation site. We constructed a gene targeting vector by introducing the neomycin resistance gene into exon 2 of a 7.5 kb genomic subclone of the mouse HL gene. Targeting was performed by electroporating 10 mg linearized vector into 10{sup 7} ES cells and selecting for 12 days with G418. 5/228 colonies (2.2%) had homologous recombination as shown by PCR screening and Southern analysis. We are microinjecting the 5 targeted clones into blastocysts to create an HL-deficient mouse. To date we have obtained two chimeras with contributions of 95% and 55% from 129, by coat color estimates. Three of 27 (11%) of the HL-deficient patients studied were suggested by genomic Southern analysis to be homozygous for large intragenic deletions. We confirmed this and defined the boundaries using exonic PCR.

  3. Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members.

    PubMed

    Thomassen, Mads; Blanco, Ana; Montagna, Marco; Hansen, Thomas V O; Pedersen, Inge S; Gutiérrez-Enríquez, Sara; Menéndez, Mireia; Fachal, Laura; Santamariña, Marta; Steffensen, Ane Y; Jønson, Lars; Agata, Simona; Whiley, Phillip; Tognazzo, Silvia; Tornero, Eva; Jensen, Uffe B; Balmaña, Judith; Kruse, Torben A; Goldgar, David E; Lázaro, Conxi; Diez, Orland; Spurdle, Amanda B; Vega, Ana

    2012-04-01

    Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of

  4. Identification of alternative splicing regulators by RNA interference in Drosophila

    PubMed Central

    Park, Jung W.; Parisky, Katherine; Celotto, Alicia M.; Reenan, Robert A.; Graveley, Brenton R.

    2004-01-01

    Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing. PMID:15492211

  5. Splicing in action: assessing disease causing sequence changes

    PubMed Central

    Baralle, D; Baralle, M

    2005-01-01

    Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited. PMID:16199547

  6. Does distance matter? Variations in alternative 3' splicing regulation.

    PubMed

    Akerman, Martin; Mandel-Gutfreund, Yael

    2007-01-01

    Alternative splicing constitutes a major mechanism creating protein diversity in humans. This diversity can result from the alternative skipping of entire exons or by alternative selection of the 5' or 3' splice sites that define the exon boundaries. In this study, we analyze the sequence and evolutionary characteristics of alternative 3' splice sites conserved between human and mouse genomes for distances ranging from 3 to 100 nucleotides. We show that alternative splicing events can be distinguished from constitutive splicing by a combination of properties which vary depending on the distance between the splice sites. Among the unique features of alternative 3' splice sites, we observed an unexpectedly high occurrence of events in which a polypyrimidine tract was found to overlap the upstream splice site. By applying a machine-learning approach, we show that we can successfully discriminate true alternative 3' splice sites from constitutive 3' splice sites. Finally, we propose that the unique features of the intron flanking alternative splice sites are indicative of a regulatory mechanism that is involved in splice site selection. We postulate that the process of splice site selection is influenced by the distance between the competitive splice sites. PMID:17704130

  7. Alternative Splice in Alternative Lice.

    PubMed

    Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P; Clark, John M; Reynolds, Stuart E; Pittendrigh, Barry R; Feil, Edward J; Urrutia, Araxi O

    2015-10-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  8. Splicing therapy for neuromuscular disease.

    PubMed

    Douglas, Andrew G L; Wood, Matthew J A

    2013-09-01

    Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision. PMID:23631896

  9. Alternative Splice in Alternative Lice

    PubMed Central

    Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P.; Clark, John M.; Reynolds, Stuart E.; Pittendrigh, Barry R.; Feil, Edward J.; Urrutia, Araxi O.

    2015-01-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  10. Alternative Splicing Signatures in RNA-seq Data: Percent Spliced in (PSI).

    PubMed

    Schafer, Sebastian; Miao, Kui; Benson, Craig C; Heinig, Matthias; Cook, Stuart A; Hubner, Norbert

    2015-01-01

    Thousands of alternative exons are spliced out of messenger RNA to increase protein diversity. High-throughput sequencing of short cDNA fragments (RNA-seq) generates a genome-wide snapshot of these post-transcriptional processes. RNA-seq reads yield insights into the regulation of alternative splicing by revealing the usage of known or unknown splice sites as well as the expression level of exons. Constitutive exons are never covered by split alignments, whereas alternative exonic parts are located within highly expressed splicing junctions. The ratio between reads including or excluding exons, also known as percent spliced in index (PSI), indicates how efficiently sequences of interest are spliced into transcripts. This protocol describes a method to calculate the PSI without prior knowledge of splicing patterns. It provides a quantitative, global assessment of exon usage that can be integrated with other tools that identify differential isoform processing. Novel, complex splicing events along a genetic locus can be visualized in an exon-centric manner and compared across conditions. PMID:26439713

  11. BRCA1 EXON 11, a CERES (composite regulatory element of splicing) element involved in splice regulation.

    PubMed

    Tammaro, Claudia; Raponi, Michela; Wilson, David I; Baralle, Diana

    2014-01-01

    Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a "silent" change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES). PMID:25056543

  12. RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.

    PubMed

    Xiong, Hui Y; Alipanahi, Babak; Lee, Leo J; Bretschneider, Hannes; Merico, Daniele; Yuen, Ryan K C; Hua, Yimin; Gueroussov, Serge; Najafabadi, Hamed S; Hughes, Timothy R; Morris, Quaid; Barash, Yoseph; Krainer, Adrian R; Jojic, Nebojsa; Scherer, Stephen W; Blencowe, Benjamin J; Frey, Brendan J

    2015-01-01

    To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine. PMID:25525159

  13. Dialing for Donors

    ERIC Educational Resources Information Center

    Schaffhauser, Dian

    2012-01-01

    When times get tough, grown children often turn to their parents for help--for some extra cash, even somewhere to stay. For colleges and universities, that role is filled by alumni donors. In 2011, with education budgets slashed across the country, giving accounted for 6.5 percent of college expenditures, according to the Council for Aid to…

  14. Vitamin D and alternative splicing of RNA

    PubMed Central

    Zhou, Rui; Chun, Rene F.; Lisse, Thomas S.; Garcia, Alejandro J.; Xu, Jianzhong; Adams, John S.; Hewison, Martin

    2014-01-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. PMID:25447737

  15. Tau exon 10 alternative splicing and tauopathies

    PubMed Central

    Liu, Fei; Gong, Cheng-Xin

    2008-01-01

    Abnormalities of microtubule-associated protein tau play a central role in neurofibrillary degeneration in several neurodegenerative disorders that collectively called tauopathies. Six isoforms of tau are expressed in adult human brain, which result from alternative splicing of pre-mRNA generated from a single tau gene. Alternative splicing of tau exon 10 results in tau isoforms containing either three or four microtubule-binding repeats (3R-tau and 4R-tau, respectively). Approximately equal levels of 3R-tau and 4R-tau are expressed in normal adult human brain, but the 3R-tau/4R-tau ratio is altered in the brains in several tauopathies. Discovery of silence mutations and intronic mutations of tau gene in some individuals with frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), which only disrupt tau exon 10 splicing but do not alter tau's primary sequence, demonstrates that dysregulation of tau exon 10 alternative splicing and consequently of 3R-tau/4R-tau balance is sufficient to cause neurodegeneration and dementia. Here, we review the gene structure, transcripts and protein isoforms of tau, followed by the regulation of exon 10 splicing that determines the expression of 3R-tau or 4R-tau. Finally, dysregulation of exon 10 splicing of tau in several tauopathies is discussed. Understanding the molecular mechanisms by which tau exon 10 splicing is regulated and how it is disrupted in tauopathies will provide new insight into the mechanisms of these tauopathies and help identify new therapeutic targets to treat these disorders. PMID:18616804

  16. Variation in alternative splicing across human tissues

    PubMed Central

    Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

    2004-01-01

    Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells. PMID:15461793

  17. The behavior of bonded doubler splices for composite sandwich panels

    NASA Technical Reports Server (NTRS)

    Zeller, T. A.; Weisahaar, T. A.

    1980-01-01

    The results of an investigation into the behavior of adhesively bonded doubler splices of two composite material sandwich panels are presented. The splices are studied from three approaches: analytical; numerical (finite elements); and experimental. Several parameters that characterize the splice are developed to determine their influence upon joint strength. These parameters are: doubler overlap length; core stiffness; laminate bending stiffness; the size of the gap between the spliced sandwich panels; and room and elevated temperatures. Similarities and contrasts between these splices and the physically similar single and double lap joints are discussed. The results of this investigation suggest several possible approaches to improving the strength of the sandwich splices.

  18. Understanding donors' motivations: a study of unrelated bone marrow donors.

    PubMed

    Switzer, G E; Dew, M A; Butterworth, V A; Simmons, R G; Schimmel, M

    1997-07-01

    Medical advances in bone marrow transplantation techniques and immunosuppressive medications have dramatically increased the number of such transplants performed each year, and consequently, the demand for bone marrow from unrelated donors. Although physiological aspects of bone marrow donation have been thoroughly investigated, very few studies have examined psychosocial factors that may impact individuals' donation decisions and outcomes. To examine one particular set of donor psychosocial issues, this study investigated motives for bone marrow donation among 343 unrelated bone marrow donors who donated through the National Marrow Donor Program. Six distinct types of donor motives were identified from open-ended questionnaire responses. Donors most frequently reported motives reflecting some awareness of both the costs (to themselves) and potential benefits (to themselves and the recipient) of donation. A desire to act in accordance with social or religious precepts, expected positive feelings about donating, empathy for the recipient, and the simple desire to help another person were also commonly cited reasons for donating. Among a series of donor background characteristics, donors' gender was the variable most strongly associated with motive type; women were most likely to cite expected positive feelings, empathy, and the desire to help someone. Central study findings indicated that donor motives predicted donors reactions to donation even after the effects of donor background characteristics (including gender) were controlled. Donors who reported exchange motives (weighing costs and benefits) and donors who reported simple (or idealized) helping motives experienced the donation as less positive in terms of higher predonation ambivalence and negative postdonation psychological reactions than did remaining donors. Donors who reported positive feeling and empathy motives had the most positive donation reactions in terms of lower ambivalence, and feeling like

  19. Structural analysis of the 5 prime flanking region of the. beta. -globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa

    SciTech Connect

    Chebloune, Y.; Pagnier, J.; Trabuchet, G.; Faure, C.; Verdier, G.; Labie, D.; Nigon, V. )

    1988-06-01

    Haplotype analysis of the {beta}-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT){sub n} and (AT){sub x}T{sub y}, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT){sub n} and (AT){sub x}T{sub y} repeats. The authors found three additional structures for (AT){sub x}T{sub y} correlating with the geographic origin of the patients. Ten other nucleotide positions, 5{prime} and 3{prime} to the (AT){sub x}T{sub y} copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5{prime} flanking region of the {beta}-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  20. Systematic screening for mutations in the 5{prime}-regulatory region of the human dopamine D{sub 1} receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder

    SciTech Connect

    Cichon, S.; Noethen, M.M.; Stoeber, G.

    1996-07-26

    A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5{prime}-flanking region of the dopamine D{sub 1} receptor (DRD1) gene. This region has previously been shown to contain a functional promoter. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5{prime}-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder. 31 refs., 3 tabs.

  1. Gene and alternative splicing annotation with AIR

    PubMed Central

    Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

    2005-01-01

    Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

  2. Phosphoregulation of Ire1 RNase splicing activity

    PubMed Central

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-01-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation. PMID:24704861

  3. Being a Living Donor: Risks

    MedlinePlus

    ... surgical risks and long term complications: Long-Term Organ Specific Donor Complications Kidney Hypertension Kidney failure Proteinuria Lung Intra- ... Vancouver Forum on the care of the live organ donor: lung, liver, pancreas, and intestine data and medical ...

  4. Donor commitment and patient needs.

    PubMed

    Bakken, R; van Walraven, A-M; Egeland, T

    2004-01-01

    The article discusses views and recommendations of the World Marrow Donor Association concerning ethical issues related to the donation of hematopoietic stem cell products with respect to recruitment, evaluation, workup, and follow-up of unrelated donors. Particular emphasis is placed upon commitment of individual donors, in particular, with respect to the needs of patients to find HLA-matched donors, who may be asked to donate stem cell and other cell products more than once for given patients. PMID:14628078

  5. Analysis of differential splicing suggests different modes of short-term splicing regulation

    PubMed Central

    Topa, Hande; Honkela, Antti

    2016-01-01

    Motivation: Alternative splicing is an important mechanism in which the regions of pre-mRNAs are differentially joined in order to form different transcript isoforms. Alternative splicing is involved in the regulation of normal physiological functions but also linked to the development of diseases such as cancer. We analyse differential expression and splicing using RNA-sequencing time series in three different settings: overall gene expression levels, absolute transcript expression levels and relative transcript expression levels. Results: Using estrogen receptor α signaling response as a model system, our Gaussian process-based test identifies genes with differential splicing and/or differentially expressed transcripts. We discover genes with consistent changes in alternative splicing independent of changes in absolute expression and genes where some transcripts change whereas others stay constant in absolute level. The results suggest classes of genes with different modes of alternative splicing regulation during the experiment. Availability and Implementation: R and Matlab codes implementing the method are available at https://github.com/PROBIC/diffsplicing. An interactive browser for viewing all model fits is available at http://users.ics.aalto.fi/hande/splicingGP/ Contact: hande.topa@helsinki.fi or antti.honkela@helsinki.fi Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307611

  6. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

    PubMed

    Ebstein, F; Textoris-Taube, K; Keller, C; Golnik, R; Vigneron, N; Van den Eynde, B J; Schuler-Thurner, B; Schadendorf, D; Lorenz, F K M; Uckert, W; Urban, S; Lehmann, A; Albrecht-Koepke, N; Janek, K; Henklein, P; Niewienda, A; Kloetzel, P M; Mishto, M

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  7. SeeSite: Characterizing Relationships between Splice Junctions and Splicing Enhancers.

    PubMed

    Lo, Christine; Kakaradov, Boyko; Lokshtanov, Daniel; Boucher, Christina

    2014-01-01

    RNA splicing is a cellular process driven by the interaction between numerous regulatory sequences and binding sites, however, such interactions have been primarily explored by laboratory methods since computational tools largely ignore the relationship between different splicing elements. Current computational methods identify either splice sites or other regulatory sequences, such as enhancers and silencers. We present a novel approach for characterizing co-occurring relationships between splice site motifs and splicing enhancers. Our approach relies on an efficient algorithm for approximately solving Consensus Sequence with Outliers , an NP-complete string clustering problem. In particular, we give an algorithm for this problem that outputs near-optimal solutions in polynomial time. To our knowledge, this is the first formulation and computational attempt for detecting co-occurring sequence elements in RNA sequence data. Further, we demonstrate that SeeSite is capable of showing that certain ESEs are preferentially associated with weaker splice sites, and that there exists a co-occurrence relationship with splice site motifs. PMID:26356335

  8. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  9. Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.

    PubMed

    Simpson, Craig G; Lewandowska, Dominika; Liney, Michele; Davidson, Diane; Chapman, Sean; Fuller, John; McNicol, Jim; Shaw, Paul; Brown, John W S

    2014-07-01

    This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence. PMID:24749484

  10. Why Minority Donors Are Needed

    MedlinePlus

    ... Español Search Register with your state as an Organ Donor Home Why Donate Becoming a Donor About Donation & ... Why Donate RELATED INFORMATION Minority Focused Grantee Publications Organ Donation Process Enrolling as a Donor Trying to Save a Life Testing for Brain ...

  11. Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.

    PubMed

    Dal Mas, Andrea; Fortugno, Paola; Donadon, Irving; Levati, Lauretta; Castiglia, Daniele; Pagani, Franco

    2015-05-01

    The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2β splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2β-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes. PMID:25665175

  12. IntSplice: prediction of the splicing consequences of intronic single-nucleotide variations in the human genome.

    PubMed

    Shibata, Akihide; Okuno, Tatsuya; Rahman, Mohammad Alinoor; Azuma, Yoshiteru; Takeda, Jun-Ichi; Masuda, Akio; Selcen, Duygu; Engel, Andrew G; Ohno, Kinji

    2016-07-01

    Precise spatiotemporal regulation of splicing is mediated by splicing cis-elements on pre-mRNA. Single-nucleotide variations (SNVs) affecting intronic cis-elements possibly compromise splicing, but no efficient tool has been available to identify them. Following an effect-size analysis of each intronic nucleotide on annotated alternative splicing, we extracted 105 parameters that could affect the strength of the splicing signals. However, we could not generate reliable support vector regression models to predict the percent-splice-in (PSI) scores for normal human tissues. Next, we generated support vector machine (SVM) models using 110 parameters to directly differentiate pathogenic SNVs in the Human Gene Mutation Database and normal SNVs in the dbSNP database, and we obtained models with a sensitivity of 0.800±0.041 (mean and s.d.) and a specificity of 0.849±0.021. Our IntSplice models were more discriminating than SVM models that we generated with Shapiro-Senapathy score and MaxEntScan::score3ss. We applied IntSplice to a naturally occurring and nine artificial intronic mutations in RAPSN causing congenital myasthenic syndrome. IntSplice correctly predicted the splicing consequences for nine of the ten mutants. We created a web service program, IntSplice (http://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice) to predict splicing-affecting SNVs at intronic positions from -50 to -3. PMID:27009626

  13. Tau Splicing and the Intricacies of Dementia

    PubMed Central

    Andreadis, Athena

    2011-01-01

    Tau is a microtubule associated protein that fulfills several functions critical for neuronal formation and health. Tau discharges its functions by producing multiple isoforms via regulated alternative splicing. These isoforms modulate tau function in normal brain by altering the domains of the protein, thereby influencing its localization, conformation and post-translational modifications and hence its availability and affinity for microtubules and other ligands. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of tau structures (neurofibrillary tangles) found in brains of dementia sufferers. More specifically, aberrations in tau splicing regulation directly cause several neurodegenerative diseases which lead to dementia. In this review, I present our cumulative knowledge of tau splicing regulation in connection with neurodegeneration and also briefly go over the still-extensive list of questions that are connected to tau (dys)function. PMID:21604267

  14. Exon circularization requires canonical splice signals.

    PubMed

    Starke, Stefan; Jost, Isabelle; Rossbach, Oliver; Schneider, Tim; Schreiner, Silke; Hung, Lee-Hsueh; Bindereif, Albrecht

    2015-01-01

    Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode. PMID:25543144

  15. Origin of Spliceosomal Introns and Alternative Splicing

    PubMed Central

    Irimia, Manuel; Roy, Scott William

    2014-01-01

    In this work we review the current knowledge on the prehistory, origins, and evolution of spliceosomal introns. First, we briefly outline the major features of the different types of introns, with particular emphasis on the nonspliceosomal self-splicing group II introns, which are widely thought to be the ancestors of spliceosomal introns. Next, we discuss the main scenarios proposed for the origin and proliferation of spliceosomal introns, an event intimately linked to eukaryogenesis. We then summarize the evidence that suggests that the last eukaryotic common ancestor (LECA) had remarkably high intron densities and many associated characteristics resembling modern intron-rich genomes. From this intron-rich LECA, the different eukaryotic lineages have taken very distinct evolutionary paths leading to profoundly diverged modern genome structures. Finally, we discuss the origins of alternative splicing and the qualitative differences in alternative splicing forms and functions across lineages. PMID:24890509

  16. The human serotonin 5-HT{sub 2C} receptor: Complete cDNA, genomic structure, and alternatively spliced variant

    SciTech Connect

    Xie, Enzhong; Zhu, Lingyu; Zhao, Lingyun

    1996-08-01

    The complete 4775-nt cDNA encoding the human serotonin 5-HT{sub 2C} receptor (5-HT{sub 2C}R), a G-protein-coupled receptor, has been isolated. It contains a 1377-nt coding region flanked by a 728-nt 5{prime}-untranslated region and a 2670-nt 3{prime}-untranslated region. By using the cloned 5-HT{sub 2C}R cDNA probe, the complete human gene for this receptor has been isolated and shown to contain six exons and five introns spanning at least 230 kb of DNA. The coding region of the human 5-HT{sub 2C}R gene is interrupted by three introns, and the positions of the intron/exon junctions are conserved between the human and the rodent genes. In addition, an alternatively spliced 5-HT{sub 2C}R RNA that contains a 95-nt deletion in the region coding for the second intracellular loop and the fourth transmembrane domain of the receptor has been identified. This deletion leads to a frameshift and premature termination so that the short isoform RNA encodes a putative protein of 248 amino acids. The ratio for the short isoform over the 5-HT{sub 2C}R RNA was found to be higher in choroid plexus tumor than in normal brain tissue, suggesting the possibility of differential regulation of the 5-HT{sub 2C}R gene in different neural tissues or during tumorigenesis. Transcription of the human 5-HT{sub 2C}R gene was found to be initiated at multiple sites. No classical TATA-box sequence was found at the appropriate location, and the 5{prime}-flanking sequence contains many potential transcription factor-binding sites. A 7.3-kb 5{prime}-flanking 5-HT{sub 2C}R DNA directed the efficient expression of a luciferase reported gene in SK-N-SH and IMR32 neuroblastoma cells, indicating that is contains a functional promoter. 69 refs., 8 figs., 1 tab.

  17. SPA: A Probabilistic Algorithm for Spliced Alignment

    PubMed Central

    van Nimwegen, Erik; Paul, Nicodeme; Sheridan, Robert; Zavolan, Mihaela

    2006-01-01

    Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5′ and 3′ ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non-canonical splice

  18. Splicing mutation in MVK is a cause of porokeratosis of Mibelli.

    PubMed

    Zeng, Kang; Zhang, Qi-Guo; Li, Li; Duan, Yan; Liang, Yan-Hua

    2014-10-01

    Porokeratosis is a chronic skin disorder characterized by the presence of patches with elevated, thick, keratotic borders, with histological cornoid lamella. Classic porokeratosis of Mibelli (PM) frequently appears in childhood with a risk of malignant transformation. Disseminated superficial actinic porokeratosis (DSAP) is the most common subtype of porokeratosis with genetic heterogeneities, and mevalonate kinase gene (MVK) mutations have been identified in minor portion of DSAP families of Chinese origin. To confirm the previous findings about MVK mutations in DSAP patients and test MVK's role(s) in PM development, we performed genomic sequence analysis for 3 DSAP families and 1 PM family of Chinese origin. We identified a splicing mutation of MVK gene, designated as c.1039+1G>A, in the PM family. No MVK mutations were found in three DSAP families. Sequence analysis for complementary DNA templates from PM lesions of all patients revealed a mutation at splice donor site of intron 10, designated as c.1039+1G>A, leading to the splicing defect and termination codon 52 amino acids after exon 10. Although no MVK mutations in DSAP patients were found as reported previously, we identified MVK simultaneously responsible for PM development. PMID:24781643

  19. Confidentiality and American semen donors.

    PubMed

    Karow, A M

    1993-01-01

    Most American donor insemination programs include a policy of complete confidentiality concerning the donor of the semen. This is the result of a long legal tradition of American constitutional law. However, some slight abridgement of this body of legal decisions might be very much in the best interests of children arising from donor insemination, and even--in most cases, in fact--the donors themselves. With regard to the children, the factors involved are both those of genetic counseling, should the need arise, and psychological development. Of course, as at present, the donor must be relieved of all responsibility, both legal and financial. PMID:8348162

  20. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  1. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2012-10-01 2012-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  2. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  3. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  4. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  5. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  6. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  7. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  8. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  9. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2013-10-01 2013-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  10. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  11. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2011-10-01 2011-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  12. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  13. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  14. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  15. Blood Donor Management in China

    PubMed Central

    Shi, Ling; Wang, Jingxing; Liu, Zhong; Stevens, Lori; Sadler, Andrew; Ness, Paul; Shan, Hua

    2014-01-01

    Summary Despite a steady increase in total blood collections and voluntary non-remunerated blood donors, China continues to have many challenges with its blood donation system. The country's donation rate remains low at 9%o, with over 60% of donors being first-time donors. Generally there is a lack of adequate public awareness about blood donation. The conservative donor selection criteria, the relatively long donation interval, and the small donation volume have further limited blood supply. To ensure a sufficient and safe blood supply that meets the increasing clinical need for blood products, there is an urgent need to strengthen the country's blood donor management. This comprehensive effort should include educating and motivating more individuals especially from the rural areas to be involved in blood donation, developing rational and evidence-based selection criteria for donor eligibility, designing a donor follow-up mechanism to encourage more future donations, assessing the current donor testing strategy, improving donor service and care, building regional and national shared donor deferral database, and enhancing the transparency of the blood donation system to gain more trust from the general public. The purpose of the review is to provide an overview of the key process of and challenges with the blood donor management system in China. PMID:25254023

  16. Blood donor management in china.

    PubMed

    Shi, Ling; Wang, Jingxing; Liu, Zhong; Stevens, Lori; Sadler, Andrew; Ness, Paul; Shan, Hua

    2014-07-01

    Despite a steady increase in total blood collections and voluntary non-remunerated blood donors, China continues to have many challenges with its blood donation system. The country's donation rate remains low at 9%o, with over 60% of donors being first-time donors. Generally there is a lack of adequate public awareness about blood donation. The conservative donor selection criteria, the relatively long donation interval, and the small donation volume have further limited blood supply. To ensure a sufficient and safe blood supply that meets the increasing clinical need for blood products, there is an urgent need to strengthen the country's blood donor management. This comprehensive effort should include educating and motivating more individuals especially from the rural areas to be involved in blood donation, developing rational and evidence-based selection criteria for donor eligibility, designing a donor follow-up mechanism to encourage more future donations, assessing the current donor testing strategy, improving donor service and care, building regional and national shared donor deferral database, and enhancing the transparency of the blood donation system to gain more trust from the general public. The purpose of the review is to provide an overview of the key process of and challenges with the blood donor management system in China. PMID:25254023

  17. Splicing is required for transactivation by the immediate early gene 1 of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

    PubMed

    Pearson, M N; Rohrmann, G F

    1997-08-18

    A region of the Lymantria disper multinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirus ie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative splice donor site and the other of which is 29% identical to AcMNPV ie-1 and contains a highly conserved splice acceptor consensus sequences. Plasmids containing the LdMNPV ORF141 and ie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing the ie-1 region alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension analysis confirmed the presence of spliced ie-0 mRNA transcripts starting at 6 hr and continuing throughout the time course of viral infection of the L dispar cell line Ld652Y. Using a plasmid containing the ie-0 spliced form of the gene as a transactivator, hr4, one of the eight homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPV hr5 enhancer did not show increased activity when cotransfected with LdMNPV ie-0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system. LdMNPV ie-0 acted as an essential replication gene, but LdMNPV ie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line. PMID:9300047

  18. Inhibition of Splicing but not Cleavage at the 5' Splice Site by Truncating Human β -globin Pre-mRNA

    NASA Astrophysics Data System (ADS)

    Furdon, Paul J.; Kole, Ryszard

    1986-02-01

    Human β -globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. All of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps--i.e., cleavage at the 3' splice site and exon ligation--need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2+ are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.

  19. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

    PubMed

    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known. PMID:26685868

  20. Histone methylation, alternative splicing and neuronal differentiation.

    PubMed

    Fiszbein, Ana; Kornblihtt, Alberto R

    2016-01-01

    Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs. PMID:27606339

  1. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  2. Development of Therapeutic Splice-Switching Oligonucleotides

    PubMed Central

    Kryczka, Adrianna; Liu, Yuqi; Badi, Yusef E.; Wong, Jessie J.; Owen, James S.; Khoo, Bernard

    2014-01-01

    Abstract Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne muscular dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase 1 clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exon-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Last, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case. PMID:24826963

  3. RNA structure in splicing: An evolutionary perspective.

    PubMed

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

    2016-09-01

    Pre-mRNA splicing is a key post-transcriptional regulation process in which introns are excised and exons are ligated together. A novel class of structured intron was recently discovered in fish. Simple expansions of complementary AC and GT dimers at opposite boundaries of an intron were found to form a bridging structure, thereby enforcing correct splice site pairing across the intron. In some fish introns, the RNA structures are strong enough to bypass the need of regulatory protein factors for splicing. Here, we discuss the prevalence and potential functions of highly structured introns. In humans, structured introns usually arise through the co-occurrence of C and G-rich repeats at intron boundaries. We explore the potentially instructive example of the HLA receptor genes. In HLA pre-mRNA, structured introns flank the exons that encode the highly polymorphic β sheet cleft, making the processing of the transcript robust to variants that disrupt splicing factor binding. While selective forces that have shaped HLA receptor are fairly atypical, numerous other highly polymorphic genes that encode receptors contain structured introns. Finally, we discuss how the elevated mutation rate associated with the simple repeats that often compose structured intron can make structured introns themselves rapidly evolving elements. PMID:27454491

  4. Electronic energy transfer and quenching in copolymers of styrene and 2-(2-prime-hydroxy -5-prime-vinylphenyl)-2H-benzotriazole Photochemical processes in polymeric systems. X

    NASA Technical Reports Server (NTRS)

    Coulter, D. R.; Gupta, A.; Yavrouian, A.; Scott, G. W.; Oconnor, D.

    1986-01-01

    Intensity and lifetime quenching of the polystyrene monomer and excimer fluorescence emission by a pendant quencher have been studied in the solid state and in solution for a series of copolymers of styrene and 2-(2-prime-hydroxy-5-prime-vinylphenyl)-2H-benzotriazole. Two mechanisms of quenching have been identified. One involves interception of the migrating monomer excitation by the quencher, and the other involves a single-step Foerster energy-transfer process from the excimer traps to a quencher. The relative efficiencies of these two quenching mechanisms in the solid state and in solution have been compared and related to the efficiency of electronic energy migration. An estimate of the excimer trap concentration in solid polystyrene is also reported.

  5. RNA splicing factors as oncoproteins and tumour suppressors.

    PubMed

    Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K

    2016-07-01

    The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these 'spliceosomal mutations' suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic and biological effects of spliceosomal mutations are crucial for the development of cancer therapies targeted at these mutations. PMID:27282250

  6. Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants

    PubMed Central

    Vallée, Maxime P.; Di Sera, Tonya L.; Nix, David A.; Paquette, Andrew M.; Parsons, Michael T.; Bell, Russel; Hoffman, Andrea; Hogervorst, Frans B. L.; Goldgar, David E.; Spurdle, Amanda B.

    2016-01-01

    ABSTRACT Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs—the “integrated evaluation”—that combines a sequence analysis‐based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis‐based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice‐site fitness program MaxEntScan to generate spliceogenicity‐based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity‐based probability of pathogenicity for BRCA gene single‐nucleotide substitutions. We also updated the BRCA gene Ex‐UV LOVD, available at http://hci‐exlovd.hci.utah.edu, with 77 re‐evaluable variants. PMID:26913838

  7. Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

    PubMed Central

    Thanaraj, T. A.; Clark, Francis

    2001-01-01

    It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF35-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms. PMID:11410667

  8. UnSplicer: mapping spliced RNA-Seq reads in compact genomes and filtering noisy splicing.

    PubMed

    Burns, Paul D; Li, Yang; Ma, Jian; Borodovsky, Mark

    2014-02-01

    Accurate mapping of spliced RNA-Seq reads to genomic DNA has been known as a challenging problem. Despite significant efforts invested in developing efficient algorithms, with the human genome as a primary focus, the best solution is still not known. A recently introduced tool, TrueSight, has demonstrated better performance compared with earlier developed algorithms such as TopHat and MapSplice. To improve detection of splice junctions, TrueSight uses information on statistical patterns of nucleotide ordering in intronic and exonic DNA. This line of research led to yet another new algorithm, UnSplicer, designed for eukaryotic species with compact genomes where functional alternative splicing is likely to be dominated by splicing noise. Genome-specific parameters of the new algorithm are generated by GeneMark-ES, an ab initio gene prediction algorithm based on unsupervised training. UnSplicer shares several components with TrueSight; the difference lies in the training strategy and the classification algorithm. We tested UnSplicer on RNA-Seq data sets of Arabidopsis thaliana, Caenorhabditis elegans, Cryptococcus neoformans and Drosophila melanogaster. We have shown that splice junctions inferred by UnSplicer are in better agreement with knowledge accumulated on these well-studied genomes than predictions made by earlier developed tools. PMID:24259430

  9. Multiple splicing pathways of group II trans-splicing introns in wheat mitochondria.

    PubMed

    Massel, Karen; Silke, Jordan R; Bonen, Linda

    2016-05-01

    Trans-splicing of discontinuous introns in plant mitochondria requires the assembly of independently-transcribed precursor RNAs into splicing-competent structures, and they are expected to be excised as Y-branched molecules ("broken lariats") because these introns belong to the group II ribozyme family. We now demonstrate that this is just one of several trans-splicing pathways for wheat mitochondrial nad1 intron 4 and nad5 intron 2; they also use a hydrolytic pathway and the liberated 5'-half-intron linear molecules are unexpectedly abundant in the RNA population. We also observe a third productive splicing pathway for nad5 intron 2 that yields full-length excised introns in which the termini are joined in vivo and possess non-encoded nucleotides. In the case of trans-splicing nad1 intron 1, which has a weakly-structured and poorly-conserved core sequence, excision appears to be solely through a hydrolytic pathway. When wheat embryos are germinated in the cold rather than at room temperature, an increased complexity in trans-splicing products is seen for nad1 intron 4, suggesting that there can be environmental effects on the RNA folding of bipartite introns. Our observations provide insights into intron evolution and the complexity of RNA processing events in plant mitochondria. PMID:26970277

  10. Regulation of alternative splicing of CD45 by antagonistic effects of SR protein splicing factors.

    PubMed

    ten Dam, G B; Zilch, C F; Wallace, D; Wieringa, B; Beverley, P C; Poels, L G; Screaton, G R

    2000-05-15

    CD45 is a transmembrane glycoprotein possessing tyrosine phosphatase activity, which is involved in cell signaling. CD45 is expressed on the surface of most leukocytes and can be alternatively spliced by the inclusion or skipping of three variable exons (4, 5, and 6 or A, B, and C) to produce up to eight isoforms. In T cells, the splicing pattern of CD45 isoforms changes after activation; naive cells express high m.w. isoforms of CD45 which predominantly express exon A (CD45RA), whereas activated cells lose expression of exon A to form low m.w. isoforms of CD45 including CD45RO. Little is known about the specific factors controlling the switch in CD45 splicing which occurs on activation. In this study, we examined the influence of the SR family of splicing factors, which, like CD45, are expressed in tissue-specific patterns and have been shown to modulate the alternative splicing of a variety of transcripts. We show that specific SR proteins have antagonistic effects on CD45 splicing, leading either to exon inclusion or skipping. Furthermore, we were able to demonstrate specific changes in the SR protein expression pattern during T cell activation. PMID:10799890

  11. Splicing factor hnRNPH drives an oncogenic splicing switch in gliomas

    PubMed Central

    LeFave, Clare V; Squatrito, Massimo; Vorlova, Sandra; Rocco, Gina L; Brennan, Cameron W; Holland, Eric C; Pan, Ying-Xian; Cartegni, Luca

    2011-01-01

    In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-α/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur d′Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16- or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness. PMID:21915099

  12. Differential regulation of alternative 3{prime} splicing of {epsilon} messenger RNA variants

    SciTech Connect

    Diaz-Sanchez, D.; Zhang, K.; Saxon, A.

    1995-08-15

    Alternative 3{prime} splicing of the one active human {epsilon} heavy chain gene results in variants of {epsilon} mRNA encoding distinct IgE proteins. The same relative amounts of these {epsilon} mRNA variants were produced by non-atopic donor B cells when driven in a variety of T-dependent or T-independent systems. The most abundant variants were those for classic secreted {epsilon} and a novel secreted form (CH4-M2{double_prime}). In contrast, cells from subjects with high levels of serum IgE secondary to parasitic infection or atopy spontaneously produced higher relative levels of the CH4-M2{prime} {epsilon} mRNA variant, lower relative amounts of both the membrane and CH4-M2{double_prime} secreted variants, and very low levels of the CH4{prime}-CH5 variant. The existence of and corresponding changes in levels of the CH4-M2{prime}-enclosed secreted protein were demonstrated. IL-10 induced this same differential expression of {epsilon} splice variants in vitro when used to costimulate IL-4 plus CD40-driven B cells and could differentially enhance the production of CH4-M2{prime} protein by established IgE-secreting cell lines. Inhibition of IgE by cross-linking the low affinity IgE receptor (CD23) decreased the levels of {epsilon} mRNA and resulted in a distinct pattern of {epsilon} mRNA characterized by a dramatic decrease in CH4-M2{prime} splice variant. IL-6, IL-2, or IFN-{gamma} did not change the {epsilon} mRNA pattern. Overall, the absolute and relative amounts of the different {epsilon} mRNA splice variants produced appear to be controlled in a differentiation-related fashion.

  13. Single Mode Fiber Optic Connectors And Splices

    NASA Astrophysics Data System (ADS)

    Woods, John G.

    1984-08-01

    There is a trend toward increasing use of single mode transmission, particularly in telecommunications where high data bit rates are transmitted for long distances. Inter-connections of multimode fibers can be made in a number of ways, using ferrules, v-grooves, elastomeric splices, etc. However, the connection of single mode fibers, which have core diameters of 4 to 13 μm, requires more precise alignment than do the multimode fibers having core diameters of 50 μm or more. At TRW, we have adapted the four rod alignment guide concept for single mode fiber inter-connections. The principle of this OPTAGUIDE* alignment guide is presented. The single mode connectors and splices use the four rod scheme with an index matching material to eliminate or reduce the losses incurred through fiber end roughness or angularity. We are able to produce demountable connectors for 80/4.4 pm fibers having typical insertion losses of 1.0dB. The main factors in obtaining this result are the naturally precise fiber alignment provided by the alignment guide, and the ability of several manufacturers to maintain tight diametral and core offset tolerances. The single mode OPTALIGN* SM Connectors have been subjected to performance and environmental tests including repeated matings, temperature cycle and vibration. The results of these tests are described in this paper. A feature of the OPTALIGN* SM Connectors is the relative ease and speed of attachment to fiber optic cable in the field, without the use of epoxy or polishing procedures. The alignment guide concept has also been applied to permanent single mode splices. The splicing procedure is simple to perform in the field without expensive or delicate equipment. Construction and assembly procedures of the demountable connectors and permanent splices will be described with the aid of diagrams and photographs.

  14. Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-1) generated by alternative splicing of the CPT-ibeta gene.

    PubMed Central

    Yu, G S; Lu, Y C; Gulick, T

    1998-01-01

    Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid beta-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (alpha) or fat and muscle (beta). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Ibeta transcript, in addition to the beta and alpha cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors. PMID:9693124

  15. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  16. IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

    PubMed

    Zheng, S

    2016-01-01

    Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. PMID:27241759

  17. Genomic features defining exonic variants that modulate splicing

    PubMed Central

    2010-01-01

    Background Single point mutations at both synonymous and non-synonymous positions within exons can have severe effects on gene function through disruption of splicing. Predicting these mutations in silico purely from the genomic sequence is difficult due to an incomplete understanding of the multiple factors that may be responsible. In addition, little is known about which computational prediction approaches, such as those involving exonic splicing enhancers and exonic splicing silencers, are most informative. Results We assessed the features of single-nucleotide genomic variants verified to cause exon skipping and compared them to a large set of coding SNPs common in the human population, which are likely to have no effect on splicing. Our findings implicate a number of features important for their ability to discriminate splice-affecting variants, including the naturally occurring density of exonic splicing enhancers and exonic splicing silencers of the exon and intronic environment, extensive changes in the number of predicted exonic splicing enhancers and exonic splicing silencers, proximity to the splice junctions and evolutionary constraint of the region surrounding the variant. By extending this approach to additional datasets, we also identified relevant features of variants that cause increased exon inclusion and ectopic splice site activation. Conclusions We identified a number of features that have statistically significant representation among exonic variants that modulate splicing. These analyses highlight putative mechanisms responsible for splicing outcome and emphasize the role of features important for exon definition. We developed a web-tool, Skippy, to score coding variants for these relevant splice-modulating features. PMID:20158892

  18. A serine–arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii

    PubMed Central

    Yeoh, Lee M.; Goodman, Christopher D.; Hall, Nathan E.; van Dooren, Giel G.; McFadden, Geoffrey I.; Ralph, Stuart A.

    2015-01-01

    Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

  19. A serine-arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii.

    PubMed

    Yeoh, Lee M; Goodman, Christopher D; Hall, Nathan E; van Dooren, Giel G; McFadden, Geoffrey I; Ralph, Stuart A

    2015-05-19

    Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine-rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

  20. Alternative splicing generates a second isoform of the catalytic A subunit of the vacuolar H(+)-ATPase.

    PubMed Central

    Hernando, N; Bartkiewicz, M; Collin-Osdoby, P; Osdoby, P; Baron, R

    1995-01-01

    We have identified a second isoform of the catalytic A subunit of the vacuolar H+ pump in chicken osteoclasts. In this isoform (A2) a 72-bp cassette replaces a 90-bp cassette present in the classical A1 isoform. The A1-specific cassette encodes a region of the protein that contains one of the three ATP-binding consensus sequences (the P-loop) identified in this polypeptide, as well as the pharmacologically relevant Cys254. In contrast, the A2-specific cassette does not contain any of these features. These two isoforms, which appear to be ubiquitously expressed, are encoded by a single gene and are generated by alternative splicing of two mutually exclusive exons. The alternative RNA processing involves the recognition of a single site, the boundary between the A2- and A1-specific exons, as either acceptor (in A1) or donor (in A2) splice site. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597085

  1. Organ Donor FAQ's: Who Can Be a Donor

    MedlinePlus

    ... citizens have been organ donors. Can non-resident aliens donate and receive organs? Non-resident aliens can both donate and receive organs in the ... the 12,375 organ donors were non-resident aliens. In this same year, 259 (1%) of the ...

  2. Introns, alternative splicing, spliced leader trans-splicing and differential expression of pcna and cyclin in Perkinsus marinus.

    PubMed

    Zhang, Huan; Dungan, Christopher F; Lin, Senjie

    2011-01-01

    To gain understanding on the structure and regulation of growth-related genes of the parasitic alveolatePerkinsus marinus, we analyzed genes encoding proliferating cell nuclear antigen (pcna) and cyclins (cyclin). Comparison of the full-length cDNAs with the corresponding genomic sequences revealedtrans-splicing of the mRNAs of these genes with a conserved 21-22 nt spliced leader. Over 10 copies ofpcnawere detected, with identical gene structures and similar nucleotide (nt) sequences (88-99%), encoding largely identical amino acid sequences (aa). Two distinct types ofcyclin(Pmacyclin1 andPmacyclin2) were identified, with 66-69% nt and 81-85% aa similarities.Pmacyclin2 was organized in tandem repeats, and was alternatively spliced, giving rise to five subtypes of transcripts. For bothpcnaandcyclingenes, 6-10 introns were found. Quantitative RT-PCR assays showed thatpcnaandPmacyclin2 expression levels were low with small variations during a 28-h time course, whereasPmacyclin1 transcript abundance was 10-100 times higher, and increased markedly during active cell division, suggesting that it is a mitoticcyclinand can be a useful growth marker for this species. The gene structure and expression features along with phylogenetic results position this organism between dinoflagellates and apicomplexans, but its definitive affiliation among alveolates requires further studies. PMID:20650682

  3. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates.

    PubMed

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex 'splicing code'. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease. PMID:19495418

  4. Evolution of Nova-Dependent Splicing Regulation in the Brain

    PubMed Central

    Živin, Marko; Darnell, Robert B

    2007-01-01

    A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs. PMID:17937501

  5. Entropic contributions to the splicing process

    NASA Astrophysics Data System (ADS)

    Osella, Matteo; Caselle, Michele

    2009-12-01

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model.

  6. Genome-wide identification and characterization of tissue-specific RNA editing events in D. melanogaster and their potential role in regulating alternative splicing

    PubMed Central

    Mazloomian, Alborz; Meyer, Irmtraud M

    2015-01-01

    RNA editing is a widespread mechanism that plays a crucial role in diversifying gene products. Its abundance and importance in regulating cellular processes were revealed using new sequencing technologies. The majority of these editing events, however, cannot be associated with regulatory mechanisms. We use tissue-specific high-throughput libraries of D. melanogaster to study RNA editing. We introduce an analysis pipeline that utilises large input data and explicitly captures ADAR's requirement for double-stranded regions. It combines probabilistic and deterministic filters and can identify RNA editing events with a low estimated false positive rate. Analyzing ten different tissue types, we predict 2879 editing sites and provide their detailed characterization. Our analysis pipeline accurately distinguishes genuine editing sites from SNPs and sequencing and mapping artifacts. Our editing sites are 3 times more likely to occur in exons with multiple splicing acceptor/donor sites than in exons with unique splice sites (p-value < 2.10−15). Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other. For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure. PMID:26512413

  7. Receptor mutations and haplotypes in growth hormone receptor deficiency: a global survey and identification of the Ecuadorean E180splice mutation in an oriental Jewish patient.

    PubMed

    Berg, M A; Peoples, R; Pérez-Jurado, L; Guevara-Aguirre, J; Rosenbloom, A L; Laron, Z; Milner, R D; Francke, U

    1994-04-01

    Eight different mutations were detected in the growth hormone (GH) receptor gene of patients with inherited GH receptor deficiency (GHRD; Laron syndrome) from five continents. All the mutations are located in the extracellular domain of the receptor and are predicted to cause gross structural abnormalities and non-functional receptor molecules. They include three nucleotide changes in the coding region causing translational stop signals, including the newly identified E183X mutation; two nucleotide changes in introns that affect splice junctions; two dinucleotide deletions that result in stop codons downstream; and one single nucleotide change that activates a donor splice site within an exon and results in a transcript missing 24 nucleotides. This latter mutation (E180splice) was first identified in a cohort of patients with GHRD from southern Ecuador. Based on the fact that the E180splice mutation generates a new cleavage site for the restriction enzyme MnlI, a simple diagnostic test has been developed that can be carried out on dried blood spots collected on filter paper. A total of 55 affected individuals from Ecuador has been found to be homozygous for this mutation. Asymptomatic carriers can also be detected, and 104 of 150 individuals screened were found to be carriers. Using this test, the E180splice mutation has recently been detected in one of two oriental Jewish patients from Israel. PMID:7949594

  8. Donor Preferences and Charitable Giving

    ERIC Educational Resources Information Center

    Williams, Stephanie Roderick

    2007-01-01

    This study aimed to learn more of the differences that may exist between the two most powerful groups of donors today, baby boomers (40-58 years old) and mature donors (59 and older), in an effort to help organizations improve fundraising efforts. Questions about the importance of organizational efficiency, program outcomes, and the desire for…

  9. Splicing variants of porcine synphilin-1.

    PubMed

    Larsen, Knud; Madsen, Lone Bruhn; Farajzadeh, Leila; Bendixen, Christian

    2015-09-01

    Parkinson's disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation. PMID:26101749

  10. Vials: Visualizing Alternative Splicing of Genes

    PubMed Central

    Strobelt, Hendrik; Alsallakh, Bilal; Botros, Joseph; Peterson, Brant; Borowsky, Mark; Pfister, Hanspeter; Lex, Alexander

    2016-01-01

    Alternative splicing is a process by which the same DNA sequence is used to assemble different proteins, called protein isoforms. Alternative splicing works by selectively omitting some of the coding regions (exons) typically associated with a gene. Detection of alternative splicing is difficult and uses a combination of advanced data acquisition methods and statistical inference. Knowledge about the abundance of isoforms is important for understanding both normal processes and diseases and to eventually improve treatment through targeted therapies. The data, however, is complex and current visualizations for isoforms are neither perceptually efficient nor scalable. To remedy this, we developed Vials, a novel visual analysis tool that enables analysts to explore the various datasets that scientists use to make judgments about isoforms: the abundance of reads associated with the coding regions of the gene, evidence for junctions, i.e., edges connecting the coding regions, and predictions of isoform frequencies. Vials is scalable as it allows for the simultaneous analysis of many samples in multiple groups. Our tool thus enables experts to (a) identify patterns of isoform abundance in groups of samples and (b) evaluate the quality of the data. We demonstrate the value of our tool in case studies using publicly available datasets. PMID:26529712

  11. Integrating alternative splicing detection into gene prediction

    PubMed Central

    Foissac, Sylvain; Schiex, Thomas

    2005-01-01

    Background Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. Results We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). Conclusions This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline. PMID:15705189

  12. Splicing of many human genes involves sites embedded within introns

    PubMed Central

    Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; Längst, Gernot; Cook, Peter R.; Papantonis, Argyris

    2015-01-01

    The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131

  13. A general role for splicing enhancers in exon definition.

    PubMed Central

    Lam, Bianca J; Hertel, Klemens J

    2002-01-01

    Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection. PMID:12403462

  14. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    PubMed Central

    Kroll, Jose E.; Kim, Jihoon; Ohno-Machado, Lucila

    2015-01-01

    Motivation. Alternative splicing events (ASEs) are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background. Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files) using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events. Availability and Implementation.Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http

  15. Superconducting cable-in-conduit low resistance splice

    DOEpatents

    Artman, Thomas A.

    2003-06-24

    A low resistance splice connects two cable-in-conduit superconductors to each other. Dividing collars for arranging sub-cable units from each conduit are provided, along with clamping collars for mating each sub-cable wire assembly to form mated assemblies. The mated assemblies ideally can be accomplished by way of splicing collar. The mated assemblies are cooled by way of a flow of coolant, preferably helium. A method for implementing such a splicing is also described.

  16. The Lombardy Rare Donor Programme

    PubMed Central

    Revelli, Nicoletta; Villa, Maria Antonietta; Paccapelo, Cinzia; Manera, Maria Cristina; Rebulla, Paolo; Migliaccio, Anna Rita; Marconi, Maurizio

    2014-01-01

    Background In 2005, the government of Lombardy, an Italian region with an ethnically varied population of approximately 9.8 million inhabitants including 250,000 blood donors, founded the Lombardy Rare Donor Programme, a regional network of 15 blood transfusion departments coordinated by the Immunohaematology Reference Laboratory of the Ca’ Granda Ospedale Maggiore Policlinico in Milan. During 2005 to 2012, Lombardy funded LORD-P with 14.1 million euros. Materials and methods During 2005–2012 the Lombardy Rare Donor Programme members developed a registry of blood donors and a bank of red blood cell units with either rare blood group phenotypes or IgA deficiency. To do this, the Immunohaematology Reference Laboratory performed extensive serological and molecular red blood cell typing in 59,738 group O or A, Rh CCDee, ccdee, ccDEE, ccDee, K− or k− donors aged 18–55 with a record of two or more blood donations, including both Caucasians and ethnic minorities. In parallel, the Immunohaematology Reference Laboratory implemented a 24/7 service of consultation, testing and distribution of rare units for anticipated or emergent transfusion needs in patients developing complex red blood cell alloimmunisation and lacking local compatible red blood cell or showing IgA deficiency. Results Red blood cell typing identified 8,747, 538 and 33 donors rare for a combination of common antigens, negative for high-frequency antigens and with a rare Rh phenotype, respectively. In June 2012, the Lombardy Rare Donor Programme frozen inventory included 1,157 red blood cell units. From March 2010 to June 2012 one IgA-deficient donor was detected among 1,941 screened donors and IgA deficiency was confirmed in four previously identified donors. From 2005 to June 2012, the Immunohaematology Reference Laboratory provided 281 complex red blood cell alloimmunisation consultations and distributed 8,008 Lombardy Rare Donor Programme red blood cell units within and outside the region

  17. Identification of RNA splicing errors resulting in human ornithine transcarbamylase deficiency.

    PubMed Central

    Carstens, R P; Fenton, W A; Rosenberg, L R

    1991-01-01

    Ornithine transcarbamylase (OTC) is an X-linked, liver-specific enzyme that catalyzes the second step of the urea cycle. In humans, inherited deficiency of OTC in hemizygous affected males usually results in severe ammonia intoxication and early death. To characterize mutations responsible for OTC deficiency, we used the PCR to amplify cDNAs prepared from patient livers which demonstrated no OTC enzyme activity and no OTC cross-reacting material on western blots. In three of seven cases, smaller than normal products were observed. Sequencing of these cDNAs revealed that two were missing exon 7 of the OTC gene and that the other was missing the first 12 bp of exon 5. Sequencing of genomic DNA from these three patients revealed that one mutant missing exon 7 had a T-to-C substitution in the 5' splice donor site of intron 7. The other mutant missing exon 7 had an A-to-G change in the third position of intron 7. It is interesting that both of these mutations resulted in skipping the preceding exon rather than in inclusion of some or all of the affected intron. In the third mutant, an A-to-T substitution was found in the 3' splice acceptor site at the end of intron 4. Here, a cryptic splice acceptor site within exon 5 was used. Northern blotting of liver RNA from these patients demonstrated (a) reduced, but significant, amounts of OTC mRNA in one of the patients who had a deleted exon 7 but (b) very little OTC mRNA in the other two patients. We propose that these point mutations, which result in aberrant splicing of the OTC pre-mRNAs, lead to OTC deficiency through either decreased efficiency of mRNA export from the nucleus to the cytosol or synthesis of enzyme subunits that are unstable and rapidly degraded. We speculate that abnormal mRNA splicing may represent a relatively common mechanism in the pathogenesis of this disease. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2035531

  18. Evolutionary Insights into RNA trans-Splicing in Vertebrates

    PubMed Central

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-01-01

    Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint. PMID:26966239

  19. RNA Splicing: Regulation and Dysregulation in the Heart.

    PubMed

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-01

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease. PMID:26846640

  20. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  1. Adenosine to Inosine editing frequency controlled by splicing efficiency.

    PubMed

    Licht, Konstantin; Kapoor, Utkarsh; Mayrhofer, Elisa; Jantsch, Michael F

    2016-07-27

    Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon-intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing. PMID:27112566

  2. Splice-switching antisense oligonucleotides as therapeutic drugs.

    PubMed

    Havens, Mallory A; Hastings, Michelle L

    2016-08-19

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

  3. Viral interactions with components of the splicing machinery.

    PubMed

    Meyer, F

    2016-01-01

    Eukaryotic genes are often interrupted by stretches of sequence with no protein coding potential or obvious function. After transcription, these interrupting sequences must be removed to give rise to the mature messenger RNA. This fundamental process is called RNA splicing and is achieved by complicated machinery made of protein and RNA that assembles around the RNA to be edited. Viruses also use RNA splicing to maximize their coding potential and economize on genetic space, and use clever strategies to manipulate the splicing machinery to their advantage. This article gives an overview of the splicing process and provides examples of viral strategies that make use of various components of the splicing system to promote their replicative cycle. Representative virus families have been selected to illustrate the interaction with various regulatory proteins and ribonucleoproteins. The unifying theme is fine regulation through protein-protein and protein-RNA interactions with the spliceosome components and associated factors to promote or prevent spliceosome assembly on given splice sites, in addition to a strong influence from cis-regulatory sequences on viral transcripts. Because there is an intimate coupling of splicing with the processes that direct mRNA biogenesis, a description of how these viruses couple the regulation of splicing with the retention or stability of mRNAs is also included. It seems that a unique balance of suppression and activation of splicing and nuclear export works optimally for each family of viruses. PMID:27571697

  4. Some characteristics of probabilistic one-sided splicing systems

    NASA Astrophysics Data System (ADS)

    Selvarajoo, Mathuri; Fong, Wan Heng; Sarmin, Nor Haniza; Turaev, Sherzod

    2013-04-01

    A theoretical model for DNA computing using the recombination behavior of DNA molecules known as asplicing system has been introduced in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings at the specific places and attaches the prefix of the first string to the suffix of the second string and the prefix of the second string to the suffix of the first string yielding the new strings. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions for splicing systems have been considered to increase the computational power of the languages generated. Recently, probabilistic splicing systems have been introduced where the probabilities are initially associated with the axioms, and the probabilities of the generated strings are computed from the probabilities of the initial strings. In this paper, some properties of probabilistic one-sided splicing systems, which are special types of probabilistic splicing systems, are investigated. We prove that probabilistic one-sided splicing systems can also increase the computational power of the languages generated.

  5. [Altruism and the donor].

    PubMed

    Langlois, A

    1991-08-01

    On December 20, 1988, the government of France passed a law to protect people who voluntarily participate in biomedical research. This article makes extensive reference to a major study, titled From Biology to Ethics, by Jean Bernard, a well-respected authority in the field of bioethics. The author looks at models proposed by Bernard, as examples for health volunteers, in particular, the blood donor and the self-experimenter. To set the tone of the article, she recalls the concept of altruism, as first proposed by Auguste Comte, then makes a linkage between his philosophy and Bernard's point of view. By trial and error, in their discussions, various ethics committees and the French State Council have agreed upon what constitutes fair compensation under the law. Unlike their Canadian counterparts, medical researchers in France have free access to volunteers who are not in perfect health--e.g., the elderly, people suffering from kidney deficiency, cirrhosis of the liver, etc.--but these "experimental subjects" receive no monetary compensation. Thus, healthy and less-than-healthy volunteers do not receive equal treatment under the law. This inequity, added to the fear of what amounts to a tax on the human body and the difficulty of ensuring just compensation, is giving rise to a great deal of uncertainty. PMID:1878857

  6. Successful use of the "unacceptable" heart donor.

    PubMed

    Menkis, A H; Novick, R J; Kostuk, W J; Pflugfelder, P W; Powell, A M; Thomson, D; McKenzie, F N

    1991-01-01

    Chronic shortage of donor organs has heightened interest in new strategies for increasing donor availability. Unacceptable hearts for transplant have previously been characterized by donor age greater than 40 years, more than 20% donor/recipient weight mismatch, ischemic time more than 4 hours, and the presence of coronary artery disease. A series of 185 consecutive orthotopic heart transplants were retrospectively examined. A significant number of donor hearts used were unacceptable by one or more of the above criteria. Our current approach is to match donors to recipients using a wide range of criteria. Donors are now accepted from any location in North America. We have accepted donors more than 55 years of age and donors weighing less than 50% of the recipient's body weight. Because of the chronic shortage of donor organs, donor criteria have been effectively liberalized, thereby increasing the donor pool without compromising the overall results of heart transplantation. PMID:2007168

  7. Thermal cracking with hydrogen donor diluent

    SciTech Connect

    Derbyshire, F.; Varghese, P.; Whitehurst, D.D.

    1983-07-26

    An improved hydrogen donor for hydrogen donor diluent cracking is provided by extraction with naphtha from the cracked product and hydrogenation by hydrogen transfer from a lower boiling hydrogen donor such as tetralin.

  8. Adult living donor liver imaging

    PubMed Central

    Cai, Larry; Yeh, Benjamin M.; Westphalen, Antonio C.; Roberts, John P.; Wang, Zhen J.

    2016-01-01

    Adult living donor liver transplantation (LDLT) is increasingly used for the treatment of end-stage liver disease. The three most commonly harvested grafts for LDLT are left lateral segment, left lobe, and right lobe grafts. The left lateral segment graft, which includes Couinaud’s segments II and III, is usually used for pediatric recipients or small size recipients. Most of the adult recipients need either a left or a right lobe graft. Whether a left or right lobe graft should be harvested from the donors depends on estimated graft and donor remnant liver volume, as well as biliary and vascular anatomy. Detailed preoperative assessment of the potential donor liver volumetrics, biliary and vascular anatomy, and liver parenchyma is vital to minimize risks to the donors and maximize benefits to the recipients. Computed tomography (CT) and magnetic resonance imaging (MRI) are currently the imaging modalities of choice in the preoperative evaluation of potential donors. This review provides an overview of key surgical considerations in LDLT that the radiologists must be aware of, and imaging findings on CT and MRI that the radiologists must convey to the surgeons when evaluating potential donors for LDLT. PMID:26912106

  9. Adult living donor liver imaging.

    PubMed

    Cai, Larry; Yeh, Benjamin M; Westphalen, Antonio C; Roberts, John P; Wang, Zhen J

    2016-01-01

    Adult living donor liver transplantation (LDLT) is increasingly used for the treatment of end-stage liver disease. The three most commonly harvested grafts for LDLT are left lateral segment, left lobe, and right lobe grafts. The left lateral segment graft, which includes Couinaud's segments II and III, is usually used for pediatric recipients or small size recipients. Most of the adult recipients need either a left or a right lobe graft. Whether a left or right lobe graft should be harvested from the donors depends on estimated graft and donor remnant liver volume, as well as biliary and vascular anatomy. Detailed preoperative assessment of the potential donor liver volumetrics, biliary and vascular anatomy, and liver parenchyma is vital to minimize risks to the donors and maximize benefits to the recipients. Computed tomography (CT) and magnetic resonance imaging (MRI) are currently the imaging modalities of choice in the preoperative evaluation of potential donors. This review provides an overview of key surgical considerations in LDLT that the radiologists must be aware of, and imaging findings on CT and MRI that the radiologists must convey to the surgeons when evaluating potential donors for LDLT. PMID:26912106

  10. Living kidney donors and ESRD.

    PubMed

    Ross, Lainie Friedman

    2015-07-01

    There are more than 325 living kidney donors who have developed end-stage renal disease and have been listed on the Organ Procurement and Transplantation Network (OPTN)/United Network for Organ Sharing (UNOS) deceased donor kidney wait list. The OPTN/UNOS database records where these kidney donors are listed and, if they donated after April 1994, where that donation occurred. These 2 locations are often not the same. In this commentary, I examine whether a national living donor registry should be created and whether transplantation centers should be notified when one of their living kidney donors develops end-stage renal disease. I consider and refute 5 potential objections to center notification. I explain that transplantation centers should look back at these cases and input data into a registry to attempt to identify patterns that could improve donor evaluation protocols. Creating a registry and mining the information it contains is, in my view, our moral and professional responsibility to future patients and the transplantation endeavor. As individuals and as a community, we need to acknowledge the many unknown risks of living kidney donation and take responsibility for identifying these risks. We then must share information about these risks, educate prospective donors about them, and attempt to minimize them. PMID:25936672

  11. The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

    SciTech Connect

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. )

    1991-01-01

    In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

  12. Characterization of radiation-induced products of thymidine 3{prime}-monophosphate and thymidylyl (3{prime}{yields}5{prime}) thymidine by high-performance liquid chromatography and laser-desorption fourier-transform mass spectrometry

    SciTech Connect

    Yoshida, H.; Hettich, R.L.

    1994-09-01

    High-performance liquid chromatography (HPLC) and laser-desorption Fourier-transform mass spectrometry (LD FTMS) have been applied for direct measurements of radiation-induced products of nucleic acid constituents containing thymidine. Laser desorption FTMS could be used for the direct detection (neither hydrolyzed nor derivatized) of X-ray-induced decomposition products of aqueous thymidine monophosphate. After these initial experiments, a variety of hydrogenated and hydroxylated thymine standards were acquired and examined by FTMS to assist in the identification of unknown radiation-induced decomposition products of thymine-containing nucleotides and dinucleotides. To extend these studies to dinucleotides, the radiation-induced products generated by the gamma radiolysis of thymidylyl (3{prime}{yields}5{prime}) thymidine (TpT) were isolated by reverse-phase HPLC and identified by LD FTMS. Thymine and thymidine 3{prime}-monophosphate were observed as the major products in this case. Several of the minor products of the HPLC profile were pooled in a single fraction and characterized simultaneously by LD FTMS. The resulting mass spectra indicated the presence of hydroxy-5,6-dihydothymidine monophosphate, 5,6-dihydrothymidine monophosphate and thymidine monophosphate, thymine glycol, hydroxy-5,6-dihydrothymine, 5-hydroxy-methyl-uracil and 5,6-dihydrothymine. The combination of HPLC purification and LD FTMS structural characterization provides a useful tool for the direct measurement of radiation-induced products of nucleotides and dinucleotides. 28 refs., 6 figs., 2 tabs.

  13. 30 CFR 75.830 - Splicing and repair of trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... including the replacement of: Insulation, semi-conductive tape, metallic shielding, and the outer jacket(s... include instructions for outer-jacket repairs and splices. (b) Splicing limitations. (1) Splicing of...

  14. 30 CFR 75.830 - Splicing and repair of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... including the replacement of: Insulation, semi-conductive tape, metallic shielding, and the outer jacket(s... include instructions for outer-jacket repairs and splices. (b) Splicing limitations. (1) Splicing of...

  15. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

    PubMed Central

    Berkers, Celia R.; de Jong, Annemieke; Schuurman, Karianne G.; Linnemann, Carsten; Meiring, Hugo D.; Janssen, Lennert; Neefjes, Jacques J.; Schumacher, Ton N. M.; Rodenko, Boris

    2015-01-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I–restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags. PMID:26401003

  16. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    PubMed Central

    Meyer, Katja; Koester, Tino; Staiger, Dorothee

    2015-01-01

    Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

  17. How to Motivate Whole Blood Donors to Become Plasma Donors

    PubMed Central

    2014-01-01

    This study tested the efficacy of interventions to recruit new plasma donors among whole blood donors. A sample of 924 donors was randomized to one of three conditions: control; information only by nurse; and information plus self-positive image message by nurse (SPI). Participants in the control condition only received a leaflet describing the plasma donation procedure. In the two experimental conditions the leaflet was explained face-to-face by a nurse. The dependent variables were the proportion of new plasma donors and the number of donations at six months. Overall, 141 (15.3%) new plasma donors were recruited at six months. There were higher proportions of new plasma donors in the two experimental conditions compared to the control condition (P < .001); the two experimental conditions did not differ. Also, compared to the control condition, those in the experimental conditions (all Ps < .001) gave plasma more often (information only by nurse:  d = .26; SPI: d = .32); the SPI intervention significantly outperformed (P < .05) the information only by nurse condition. The results suggest that references to feelings of SPI such as feeling good and being proud and that giving plasma is a rewarding personal experience favor a higher frequency of plasma donation. PMID:25530909

  18. A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming.

    PubMed

    Michaux, Alexandre; Larrieu, Pierre; Stroobant, Vincent; Fonteneau, Jean-François; Jotereau, Francine; Van den Eynde, Benoît J; Moreau-Aubry, Agnès; Vigneron, Nathalie

    2014-02-15

    Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work. PMID:24453253

  19. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

    Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exon–intron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing

  20. An artificial riboswitch for controlling pre-mRNA splicing.

    PubMed

    Kim, Dong-Suk; Gusti, Veronica; Pillai, Sailesh G; Gaur, Rajesh K

    2005-11-01

    Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant. PMID:16244133

  1. Modulation of RNA splicing as a potential treatment for cancer.

    PubMed

    Bauman, John A; Kole, Ryszard

    2011-01-01

    Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant.  This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ∆7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.  PMID:21637003

  2. Alternative Splicing of TAF6: Downstream Transcriptome Impacts and Upstream RNA Splice Control Elements

    PubMed Central

    Kamtchueng, Catherine; Stébenne, Marie-Éve; Delannoy, Aurélie; Wilhelm, Emmanuelle; Léger, Hélène; Benecke, Arndt G.; Bell, Brendan

    2014-01-01

    The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ. PMID:25025302

  3. Donor states in inverse opals

    NASA Astrophysics Data System (ADS)

    Mahan, G. D.

    2014-09-01

    We calculate the binding energy of an electron bound to a donor in a semiconductor inverse opal. Inverse opals have two kinds of cavities, which we call octahedral and tetrahedral, according to their group symmetry. We put the donor in the center of each of these two cavities and obtain the binding energy. The binding energies become very large when the inverse opal is made from templates with small spheres. For spheres less than 50 nm in diameter, the donor binding can increase to several times its unconfined value. Then electrons become tightly bound to the donor and are unlikely to be thermally activated to the semiconductor conduction band. This conclusion suggests that inverse opals will be poor conductors.

  4. Donor states in inverse opals

    SciTech Connect

    Mahan, G. D.

    2014-09-21

    We calculate the binding energy of an electron bound to a donor in a semiconductor inverse opal. Inverse opals have two kinds of cavities, which we call octahedral and tetrahedral, according to their group symmetry. We put the donor in the center of each of these two cavities and obtain the binding energy. The binding energies become very large when the inverse opal is made from templates with small spheres. For spheres less than 50 nm in diameter, the donor binding can increase to several times its unconfined value. Then electrons become tightly bound to the donor and are unlikely to be thermally activated to the semiconductor conduction band. This conclusion suggests that inverse opals will be poor conductors.

  5. Being a Living Donor: Risks

    MedlinePlus

    ... for blood transfusions side effects associated with allergic reactions to the anesthesia death The best source of information about risks and expected donor outcomes is your transplant team. In addition, it’s important to take an active role in ...

  6. A case of mild CHARGE syndrome associated with a splice site mutation in CHD7.

    PubMed

    Wells, Constance; Loundon, Natalie; Garabedian, Noël; Wiener-Vacher, Sylvette; Cordier-Bouvier, Marie-Dominique; Goudeffroye, Géraldine; Attié-Bitach, Tania; Marlin, Sandrine

    2016-04-01

    CHARGE syndrome (MIM#214800) (Coloboma, Heart defect, Atresia of choanae, Retarded growth and development, Genital hypoplasia, Ear abnormalities/deafness) is caused by heterozygous mutation of CHD7 transmitted in an autosomal dominant manner. In this report, we describe a patient with bilateral hearing impairment, unusually-shaped ears, no intellectual disability and a patent ductus arteriosus. Further investigation showed abnormal semicircular canals and the presence of olfactory bulbs. He does not fulfill the Blake or the Verloes criteria for CHARGE. A de novo mutation at the donor splice site of intron 33 was identified (c.7164 + 1G > A). It is of importance to diagnose mildly affected patients for appropriate genetic counselling and to better understand the mild end of the phenotypic spectrum of CHARGE syndrome. PMID:26921530

  7. Motivations for Giving of Alumni Donors, Lapsed Donors and Non-Donors: Implications for Christian Higher Education

    ERIC Educational Resources Information Center

    Rugano, Emilio Kariuki

    2011-01-01

    This descriptive and causal comparative study sought to identify motivations for alumni donor acquisition and retention in Christian institutions of higher learning. To meet this objective, motivations for alumni donors, lapsed donors, and non-donors were analyzed and compared. Data was collected through an electronic survey of a stratified sample…

  8. Regulation of alternative splicing of liver scavenger receptor class B gene by estrogen and the involved regulatory splicing factors.

    PubMed

    Zhang, Xiaohui; Moor, Andrea N; Merkler, Kathleen A; Liu, Qiyuan; McLean, Mark P

    2007-11-01

    The scavenger receptor class B isoforms (SR-B) type I and type II mediate the selective uptake of high-density lipoprotein cholesterol and promote reverse cholesterol transport, an important atherosclerosis protection mechanism, in the liver. Previously it was shown that the hepatic expression of SR-BI and SR-BII is regulated by estrogen. In the present study, we demonstrate that estrogen differentially regulates expression of the glycosylated and nonglycosylated forms of SR-BI and SR-BII in rat liver and hepatic cells. We report that estrogen mainly induces the down-regulation of glycosylated SR-BI and the up-regulation of nonglycosylated SR-BII. To study how estrogen regulates expression of the SR-B isoforms, we constructed a SR-B minigene containing minimal genomic sequences and were able to demonstrate that estrogen directly regulates the pre-mRNA alternative splicing of the exogenously expressed SR-B minigene in hepatic cells. Furthermore, we showed that the overexpression of splicing factors alternative splicing factor/splicing factor 2, Transformer (Tra)-2alpha, and Tra2beta changes the splicing pattern of SR-B dramatically, whereas other splicing factors, such as heterogeneous nuclear ribonucleoprotein-G, SC-35, and arginine/serine-rich p40, had no effect. We also demonstrate that estrogen regulates Tra2beta expression levels in liver cells. These studies suggest that estrogen may regulate SR-B isoform expression at both the RNA splicing and posttranslational modification levels and that, for alternative splicing regulation, estrogen may function by regulating the expression of the splicing factors alternative splicing factor/splicing factor 2, Tra2alpha, and especially Tra2beta. PMID:17673517

  9. Unusual Phenotypic Features in a Patient with a Novel Splice Mutation in the GHRHR Gene

    PubMed Central

    Hilal, Latifa; Hajaji, Yassir; Vie-Luton, Marie-Pierre; Ajaltouni, Zeina; Benazzouz, Bouchra; Chana, Maha; Chraïbi, Adelmajid; Kadiri, Abdelkrim; Amselem, Serge; Sobrier, Marie-Laure

    2008-01-01

    Isolated growth hormone deficiency (IGHD) may be of genetic origin. One of the few genes involved in that condition encodes the growth hormone releasing hormone receptor (GHRHR) that, through its ligand (GHRH), plays a pivotal role in the GH synthesis and secretion by the pituitary. Our objective is to describe the phenotype of two siblings born to a consanguineous union presenting with short stature (IGHD) and Magnetic Resonance Imaging (MRI) abnormalities, and to identify the molecular basis of this condition. Our main outcome measures were clinical and endocrinological investigations, MRI of the pituitary region, study of the GHRHR gene sequence and transcripts. In both patients, the severe growth retardation (−5SD) was combined with anterior pituitary hypoplasia. In addition to these classical phenotypic features for IGHD, one of the patients had a Chiari I malformation, an arachnoid cyst, and a dysmorphic anterior pituitary. A homozygous sequence variation in the consensus donor splice site of intron 1 (IVS1 + 2T > G) of the GHRHR gene was identified in both patients. Using in vitro transcription assay, we showed that this mutation results in abnormal splicing of GHRHR transcripts. In this report, which broadens the phenotype associated with GHRHR defects, we discuss the possible role of the GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GH deficiency. PMID:18297129

  10. Unusual phenotypic features in a patient with a novel splice mutation in the GHRHR gene.

    PubMed

    Hilal, Latifa; Hajaji, Yassir; Vie-Luton, Marie-Pierre; Ajaltouni, Zeina; Benazzouz, Bouchra; Chana, Maha; Chraïbi, Adelmajid; Kadiri, Abdelkrim; Amselem, Serge; Sobrier, Marie-Laure

    2008-01-01

    Isolated growth hormone deficiency (IGHD) may be of genetic origin. One of the few genes involved in that condition encodes the growth hormone releasing hormone receptor (GHRHR) that, through its ligand (GHRH), plays a pivotal role in the GH synthesis and secretion by the pituitary. Our objective is to describe the phenotype of two siblings born to a consanguineous union presenting with short stature (IGHD) and Magnetic Resonance Imaging (MRI) abnormalities, and to identify the molecular basis of this condition. Our main outcome measures were clinical and endocrinological investigations, MRI of the pituitary region, study of the GHRHR gene sequence and transcripts. In both patients, the severe growth retardation (-5SD) was combined with anterior pituitary hypoplasia. In addition to these classical phenotypic features for IGHD, one of the patients had a Chiari I malformation, an arachnoid cyst, and a dysmorphic anterior pituitary. A homozygous sequence variation in the consensus donor splice site of intron 1 (IVS1 + 2T > G) of the GHRHR gene was identified in both patients. Using in vitro transcription assay, we showed that this mutation results in abnormal splicing of GHRHR transcripts. In this report, which broadens the phenotype associated with GHRHR defects, we discuss the possible role of the GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GH deficiency. PMID:18297129

  11. A 3' splice site mutation in the thyroglobulin gene responsible for congenital goiter with hypothyroidism.

    PubMed

    Ieiri, T; Cochaux, P; Targovnik, H M; Suzuki, M; Shimoda, S; Perret, J; Vassart, G

    1991-12-01

    A case of congenital goiter with defective thyroglobulin synthesis has been studied in molecular terms. The patient is the fifth of a kindred of six, three of which have a goiter. The parents are first cousins. Segregation of thyroglobulin alleles in the family was studied by Southern blotting with a probe revealing a diallelic restriction fragment length polymorphism (RFLP). The results demonstrated that the three affected siblings were homozygous for the RFLP. Northern blotting analysis of the goiter RNA with a thyroglobulin probe suggested that thyroglobulin mRNA size was slightly reduced. Polymerase chain reaction amplification of the 8.5-kb thyroglobulin mRNA as overlapping cDNA fragments demonstrated that a 200-bp segment was missing from the 5' region of the goiter mRNA. Subcloning and sequencing of the cDNA fragments, and of the patient genomic DNA amplified from this region, revealed that exon 4 is missing from the major thyroglobulin transcript in the goiter, and that this aberrant splicing is due to a C to G transversion at position minus 3 in the acceptor splice site of intron 3. The presence in exon 4 of a putative donor tyrosine residue (Tyrosine nr 130) involved in thyroid hormone formation provides a coherent explanation to the hypothyroid status of the patient. PMID:1752952

  12. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  13. Identification of a new splice variant of BDNF in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brain-derived neurotrophic factor (BDNF) appears to be involved in the central regulation of energy homeostasis. BDNF splicing variants were discovered in vertebrates. Results from human, mouse and rat suggest that alternative BDNF splicing variants potentially play a role in fat deposition. Using t...

  14. Alternative splicing of inner-ear-expressed genes.

    PubMed

    Wang, Yanfei; Liu, Yueyue; Nie, Hongyun; Ma, Xin; Xu, Zhigang

    2016-09-01

    Alternative splicing plays a fundamental role in the development and physiological function of the inner ear. Inner-ear-specific gene splicing is necessary to establish the identity and maintain the function of the inner ear. For example, exon 68 of Cadherin 23 (Cdh23) gene is subject to inner-ear-specific alternative splicing, and as a result, Cdh23(+ 68) is only expressed in inner ear hair cells. Alternative splicing along the tonotopic axis of the cochlea contributes to frequency tuning, particularly in lower vertebrates, such as chickens and turtles. Differential splicing of Kcnma1, which encodes for the α subunit of the Ca(2+)-activated K(+) channel (BK channel), has been suggested to affect the channel gating properties and is important for frequency tuning. Consequently, deficits in alternative splicing have been shown to cause hearing loss, as we can observe in Bronx Waltzer (bv) mice and Sfswap mutant mice. Despite the advances in this field, the regulation of alternative splicing in the inner ear remains elusive. Further investigation is also needed to clarify the mechanism of hearing loss caused by alternative splicing deficits. PMID:27376950

  15. Alternative Splicing of Pre-mRNA in Cancer

    PubMed Central

    Körner, Meike; Miller, Laurence J.

    2009-01-01

    Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications. PMID:19574427

  16. Involvement of PARP1 in the regulation of alternative splicing

    PubMed Central

    Matveeva, Elena; Maiorano, John; Zhang, Qingyang; Eteleeb, Abdallah M; Convertini, Paolo; Chen, Jing; Infantino, Vittoria; Stamm, Stefan; Wang, Jiping; Rouchka, Eric C; Fondufe-Mittendorf, Yvonne N

    2016-01-01

    Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes. PMID:27462443

  17. Global analysis of alternative splicing differences between humans and chimpanzees.

    PubMed

    Calarco, John A; Xing, Yi; Cáceres, Mario; Calarco, Joseph P; Xiao, Xinshu; Pan, Qun; Lee, Christopher; Preuss, Todd M; Blencowe, Benjamin J

    2007-11-15

    Alternative splicing is a powerful mechanism affording extensive proteomic and regulatory diversity from a limited repertoire of genes. However, the extent to which alternative splicing has contributed to the evolution of primate species-specific characteristics has not been assessed previously. Using comparative genomics and quantitative microarray profiling, we performed the first global analysis of alternative splicing differences between humans and chimpanzees. Surprisingly, 6%-8% of profiled orthologous exons display pronounced splicing level differences in the corresponding tissues from the two species. Little overlap is observed between the genes associated with alternative splicing differences and the genes that display steady-state transcript level differences, indicating that these layers of regulation have evolved rapidly to affect distinct subsets of genes in humans and chimpanzees. The alternative splicing differences we detected are predicted to affect diverse functions including gene expression, signal transduction, cell death, immune defense, and susceptibility to diseases. Differences in expression at the protein level of the major splice variant of Glutathione S-transferase omega-2 (GSTO2), which functions in the protection against oxidative stress and is associated with human aging-related diseases, suggests that this enzyme is less active in human cells compared with chimpanzee cells. The results of this study thus support an important role for alternative splicing in establishing differences between humans and chimpanzees. PMID:17978102

  18. Discovering Transcription and Splicing Networks in Myelodysplastic Syndromes

    PubMed Central

    Wang, Hongyan; Wen, Jianguo; Chang, Chung-che; Zhou, Xiaobo

    2013-01-01

    More and more transcription factors and their motifs have been reported and linked to specific gene expression levels. However, focusing only on transcription is not sufficient for mechanism research. Most genes, especially in eukaryotes, are alternatively spliced to different isoforms. Some of these isoforms increase the biodiversity of proteins. From this viewpoint, transcription and splicing are two of important mechanisms to modulate expression levels of isoforms. To integrate these two kinds of regulation, we built a linear regression model to select a subset of transcription factors and splicing factors for each co-expressed isoforms using least-angle regression approach. Then, we applied this method to investigate the mechanism of myelodysplastic syndromes (MDS), a precursor lesion of acute myeloid leukemia. Results suggested that expression levels of most isoforms were regulated by a set of selected regulatory factors. Some of the detected factors, such as EGR1 and STAT family, are highly correlated with progression of MDS. We discovered that the splicing factor SRSF11 experienced alternative splicing switch, and in turn induced different amino acid sequences between MDS and controls. This splicing switch causes two different splicing mechanisms. Polymerase Chain Reaction experiments also confirmed that one of its isoforms was over-expressed in MDS. We analyzed the regulatory networks constructed from the co-expressed isoforms and their regulatory factors in MDS. Many of these networks were enriched in the herpes simplex infection pathway which involves many splicing factors, and pathways in cancers and acute or chronic myeloid leukemia. PMID:24244432

  19. Splice Form Dependence of b-Neurexin/Neuroligin Binding Interactions

    SciTech Connect

    Koehnke, J.; Katsamba, P; Ahlsen, G; Bahna, F; Vendome, J; Honig, B; Shapiro, L; Jin, X

    2010-01-01

    Alternatively spliced {beta}-neurexins ({beta}-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a {beta}-NRX/NL synaptic recognition code. We utilized surface plasmon resonance to measure binding affinities between all combinations of alternatively spliced {beta}-NRX 1-3 and NL 1-3 ectodomains. Binding was observed for all {beta}-NRX/NL pairs. The presence of the NL1 B splice insertion lowers {beta}-NRX binding affinity by 2-fold, while {beta}-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated {beta}-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new {beta} strand that replaces the {beta}10 strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in {beta}-NRX/NL-mediated synaptic recognition.

  20. Functional impact of splice isoform diversity in individual cells

    PubMed Central

    Yap, Karen; Makeyev, Eugene V.

    2016-01-01

    Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755

  1. Should pharmacologists care about alternative splicing? IUPHAR Review 4

    PubMed Central

    Bonner, T I

    2014-01-01

    Alternative splicing of mRNAs occurs in the majority of human genes, and most differential splicing results in different protein isoforms with possibly different functional properties. However, there are many reported splicing variations that may be quite rare, and not all combinatorially possible variants of a given gene are expressed at significant levels. Genes of interest to pharmacologists are frequently expressed at such low levels that they are not adequately represented in genome-wide studies of transcription. In single-gene studies, data are commonly available on the relative abundance and functional significance of individual alternatively spliced exons, but there are rarely data that quantitate the relative abundance of full-length transcripts and define which combinations of exons are significant. A number of criteria for judging the significance of splice variants and suggestions for their nomenclature are discussed. PMID:24670145

  2. Detecting Image Splicing Using Merged Features in Chroma Space

    PubMed Central

    Liu, Guangjie; Dai, Yuewei

    2014-01-01

    Image splicing is an image editing method to copy a part of an image and paste it onto another image, and it is commonly followed by postprocessing such as local/global blurring, compression, and resizing. To detect this kind of forgery, the image rich models, a feature set successfully used in the steganalysis is evaluated on the splicing image dataset at first, and the dominant submodel is selected as the first kind of feature. The selected feature and the DCT Markov features are used together to detect splicing forgery in the chroma channel, which is convinced effective in splicing detection. The experimental results indicate that the proposed method can detect splicing forgeries with lower error rate compared to the previous literature. PMID:24574877

  3. Co-transcriptional commitment to alternative splice site selection.

    PubMed

    Roberts, G C; Gooding, C; Mak, H Y; Proudfoot, N J; Smith, C W

    1998-12-15

    Production of mRNA in eukaryotic cells involves not only transcription but also various processing reactions such as splicing. Recent experiments have indicated that there are direct physical connections between components of the transcription and processing machinery, supporting previous suggestions that pre-mRNA splicing occurs co-transcriptionally. Here we have used a novel functional approach to demonstrate co-transcriptional regulation of alternative splicing. Exon 3 of the alpha-tropomyosin gene is specifically repressed in smooth muscle cells. By delaying synthesis of an essential downstream inhibitory element, we show that the decision to splice or repress exon 3 occurs during a limited window of opportunity following transcription, indicating that splice site selection proceeds rapidly after transcription. PMID:9837984

  4. The evolutionary landscape of alternative splicing in vertebrate species.

    PubMed

    Barbosa-Morais, Nuno L; Irimia, Manuel; Pan, Qun; Xiong, Hui Y; Gueroussov, Serge; Lee, Leo J; Slobodeniuc, Valentina; Kutter, Claudia; Watt, Stephen; Colak, Recep; Kim, TaeHyung; Misquitta-Ali, Christine M; Wilson, Michael D; Kim, Philip M; Odom, Duncan T; Frey, Brendan J; Blencowe, Benjamin J

    2012-12-21

    How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species. PMID:23258890

  5. Splice Resistance Measurements in 2G YBCO Coated Conductor

    SciTech Connect

    Rey, Christopher M; Duckworth, Robert C; Zhang, Yifei

    2009-01-01

    Abstract The Oak Ridge National Laboratory has been investigating the electrical splice resistance of second-generation (2G) YBCO coated conductor. The purpose of the experimental investigation is to study the splice resistance of 2G YBCO coated conductor as a function of: a) operating temperature, b) magnetic field strength (B-field), and c) magnetic field orientation ( ). Understanding the splice resistance with its corresponding variation as a function of surface preparation and operating conditions is essential to the practical implementation of electric utility devices; e.g., motors, generators, transformers, cables, and fault-current limiters, etc. Preliminary test results indicate that the 2G YBCO splice resistance shows a weak temperature dependence and a significantly stronger dependence upon magnetic field strength and magnetic field orientation. Surface preparation conditions are also briefly discussed. Index Terms coated conductor, splice, critical current, YBCO

  6. Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms

    PubMed Central

    Erkelenz, Steffen; Mueller, William F.; Evans, Melanie S.; Busch, Anke; Schöneweis, Katrin; Hertel, Klemens J.; Schaal, Heiner

    2013-01-01

    Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5′ splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection. PMID:23175589

  7. Analysis of Donor Motivations in Living Donor Liver Transplantation

    PubMed Central

    Abdeldayem, Hesham; Kashkoush, Samy; Hegab, Bassem Soliman; Aziz, Amr; Shoreem, Hany; Saleh, Shereef

    2014-01-01

    Objectives: The introduction of the living donor liver transplantation (LDLT) in Egypt as in elsewhere, has raised important psychological conflicts and ethical questions. The objective of this study was to get better understanding of the potential donors’ motives toward LDLT. Methods: This study was conducted on consecutive 193 living-liver donors who underwent partial hepatectomy as donors for LDLT during the period between April 2003 and January 2013, at the National Liver Institute Menoufeyia University, Egypt. Potential donors were thoroughly evaluated preoperatively through a screening questionnaire and interviews as regard their demographic data, relationship to the potential recipient, and motives toward proceeding to surgery. They were assured that the information shared between them and the transplant center is confidential. Results: The donors’ mean age was 25.53 ± 6.39 years with a range of 18–45 years. Males represented 64.7% and females were 35.3%. The most common donors (32.1%, n = 62) were sons and daughters to their parents (sons: n = 43, daughters: n = 19) while parents to their offsprings represent 15% (mothers: n = 21, fathers: n = 8). Brothers and sisters represent 16.5% (brothers: n = 22, sisters: n = 10). Nephews and nieces giving their uncles or aunts were 14%. The number of wives donating to their husbands was 11 (5.7%). Interestingly, there was no single husband who donated his wife. Among the remaining donors, there were 11 cousins and 1 uncle. Unrelated donors were 20 (10.4%). Several factors seemed to contribute to motivation for donation: the seriousness of the potential recipient condition, the relationship and personal history of the donor to the potential recipient, the religious beliefs, the trust in the health care system, and family dynamics and obligations. Conclusion: Absolute absence of coercion on the living-liver donor’s motives may not be realistic because of the serious

  8. A novel SMARCAL1 missense mutation that affects splicing in a severely affected Schimke immunoosseous dysplasia patient.

    PubMed

    Barraza-García, Jimena; Rivera-Pedroza, Carlos I; Belinchón, Alberta; Fernández-Camblor, Carlota; Valenciano-Fuente, Blanca; Lapunzina, Pablo; Heath, Karen E

    2016-08-01

    Schimke immunoosseous dysplasia (SIOD) is an autosomal recessive disease characterized by skeletal dysplasia, focal segmental glomerulosclerosis, renal failure and immunodeficiency. In this work, we report the molecular studies undertaken in a severely affected SIOD patient that died at six years old due to nephropathy. The patient was screened for mutations using a targeted skeletal dysplasias panel. A homozygous novel missense mutation was identified, c.1615C > G (p.[Leu539Val]) that was predicted as mildly pathogenic by in silico pathogenicity prediction tools. However, splicing prediction software suggested that this variant may create a new splicing donor site in exon 9, which was subsequently confirmed using a minigene assay in HEK293 cells. Thus, the splicing alteration, c.1615C > G; r.1615c > g, 1615_1644del; (p.[Leu539_Ile548del]), results in the loss of 10 amino acids of the HARP-ATPase catalytic domain and the RPA-binding domain. Several studies have demonstrated a weak genotype-phenotype correlation among such patients. Thus, the molecular characterization has helped us to understand why a predicted weakly pathogenic missense mutation results in severe SIOD and should be considered in similar scenarios. PMID:27282802

  9. 5 prime -Azido-(3,6- sup 3 H sub 2 )-1-naphthylphthalamic acid, a photoactivatable probe for naphthylphthalamic acid receptor proteins from higher plants: Identification of a 23-kDa protein from maize coleoptile plasma membranes

    SciTech Connect

    Zettl, R.; Feldwisch, J.; Schell, J.; Palme, K. ); Boland, W. )

    1992-01-15

    1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carrier mediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, the authors have synthesized a tritiated, photolabile NPA analogue, 5{prime}-azido-(3,6-{sup 3}H{sub 2})NPA (({sup 3}H{sub 2})N{sub 3}NPA). This analogue was used to identify NPA-binding proteins in fractions highly enriched for plasma membrane vesicles isolated from maize coleoptiles (Zea mays L.). Competition studies showed that binding of ({sup 3}H{sub 2})N{sub 3}NPA to maize plasma membrane vesicles was blocked by nonradioactive NPA but not by benzoic acid. After incubation of plasma membrane vesicles with ({sup 3}H{sub 2})N{sub 3}NPA and exposure to UV light, they observed specific photoaffinity labeling of a protein with an apparent molecular mass of 23 kDa. Pretreatment of the plasma membrane vesicles with indole-3-acetic acid or with the auxin-transport inhibitors NPA and 2,3,5-triiodobenzoic acid strongly reduced specific labeling of this protein. This 23-kDa protein was also labeled by addition of 5-azido-(7-{sup 3}H)indole-3-acetic acid to plasma membranes prior to exposure to UV light. The 23-kDa protein was solubilized from plasma membranes by 1% Triton X-100. The possibility that this 23-kDa polypeptide is part of the auxin efflux carrier system is discussed.

  10. Mechanisms and Regulation of Alternative Pre-mRNA Splicing

    PubMed Central

    Lee, Yeon

    2015-01-01

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

  11. Blood Donation by Elderly Repeat Blood Donors

    PubMed Central

    Zeiler, Thomas; Lander-Kox, Jutta; Alt, Timo

    2014-01-01

    Summary Background Upper age limits for blood donors are intended to protect elderly blood donors from donor reactions. However, due to a lack of data about adverse reactions in elderly blood donors, upper age limits are arbitrary and vary considerably between different countries. Methods Here we present data from 171,231 voluntary repeat whole blood donors beyond the age of 68 years. Results Blood donations from repeat blood donors beyond the age of 68 years increased from 2,114 in 2005 to 38,432 in 2012 (from 0,2% to 4.2% of all whole blood donations). Adverse donor reactions in repeat donors decreased with age and were lower than in the whole group (0.26%), even in donors older than 71 years (0.16%). However, from the age of 68 years, the time to complete recovery after donor reactions increased. Donor deferrals were highest in young blood donors (21.4%), but increased again in elderly blood donors beyond 71 years (12.6%). Conclusion Blood donation by regular repeat blood donors older than 71 years may be safely continued. However, due to a lack of data for donors older than 75 years, blood donation in these donors should be handled with great caution. PMID:25254019

  12. Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells

    PubMed Central

    Turton, Keren B.; Annis, Douglas S.; Rui, Lixin; Esnault, Stephane; Mosher, Deane F.

    2015-01-01

    Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3α or in STAT3β, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (ΔS). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ΔS/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-α, ΔS-α, S-β, and ΔS-β) rather than the three reported in publically available databases from which ΔS-β is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-α was the most abundant, followed by S-β. ΔS-α and ΔS-β together comprised 15.6±4.0 % (mean±SD, n=21) of the total. The percentage of STAT3β variants that were ΔS was 1.5-fold greater than of STAT3α variants that were ΔS. Inspection of Illumina’s “BodyMap” RNA-Seq database revealed that the ΔS variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-α, ΔS-α, S-β, and ΔS-β variants of STAT3. PMID:25984943

  13. WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo

    SciTech Connect

    Markus, M. Andrea; Heinrich, Bettina; Raitskin, Oleg; Adams, David J.; Mangs, Helena; Goy, Christine; Ladomery, Michael; Sperling, Ruth; Stamm, Stefan; Morris, Brian J. . E-mail: brianm@medsci.usyd.edu.au

    2006-10-15

    Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.

  14. Donor Hemovigilance with Blood Donation

    PubMed Central

    Diekamp, Ulrich; Gneißl, Johannes; Rabe, Angela; Kießig, Stephan T.

    2015-01-01

    Background Reports on unexpected events (UEs) during blood donation (BD) inadequately consider the role of technical UEs. Methods Defined local and systemic UEs were graded by severity; technical UEs were not graded. On January 1, 2008, E.B.P.S.-Logistics (EBPS) installed the UE module for plasma management software (PMS). Donor room physicians entered UEs daily into PMS. Medical directors reviewed entries quarterly. EBPS compiled data on donors, donations, and UEs from January 1, 2008 to June 30, 2011. Results 6,605 UEs were observed during 166,650 BDs from 57,622 donors for a corrected incidence of 4.30% (0.66% local, 1.59% systemic, 2.04% technical UEs). 2.96% of BDs were accompanied by one UE and 0.45% by >1 UE (2-4). 6.3% of donors donating blood for their first time, 3.5% of those giving blood for their second time, and 1.9% of donors giving their third or more BD experienced UEs. Most common UEs were: discontinued collections due to venous access problems, repeated venipuncture, and small hematomas. Severe circulatory UEs occurred at a rate of 16 per 100,000 BDs. Conclusions Technical UEs were common during BD. UEs accompanied first and second donations significantly more often than subsequent donations. PMID:26195932

  15. In vitro Splicing of Influenza Viral NS1 mRNA and NS1-β -globin Chimeras: Possible Mechanisms for the Control of Viral mRNA Splicing

    NASA Astrophysics Data System (ADS)

    Plotch, Stephen J.; Krug, Robert M.

    1986-08-01

    In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human β -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and β -globin sequences, we show that the NS1 5' splice site was effectively utilized by the β -globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the β -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from β -globin With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.

  16. Splicing analysis of unclassified variants in COL2A1 and COL11A1 identifies deep intronic pathogenic mutations

    PubMed Central

    Richards, Allan J; McNinch, Annie; Whittaker, Joanne; Treacy, Becky; Oakhill, Kim; Poulson, Arabella; Snead, Martin P

    2012-01-01

    UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5′ and 3′ of each exon, typically 30 bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patient's cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype. PMID:22189268

  17. Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations.

    PubMed

    Ramalho, Anabela S; Clarke, Luka A; Sousa, Marisa; Felicio, Verónica; Barreto, Celeste; Lopes, Carlos; Amaral, Margarida D

    2016-01-01

    The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl- channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis. PMID:25735457

  18. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    SciTech Connect

    Ris-Stalpers, C.; Kuiper, G.G.J.M.; Faber, P.W.; van Rooij, H.C.J.; Degenhart, H.J.; Trapman, J.; Brinkmann, A.O. ); Schweikert, H.U. ); Zegers, N.D. ); Hodgins, M.B. )

    1990-10-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G {r arrow} T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein {approx}5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

  19. [Living donor transplantation. Surgical complications].

    PubMed

    Karam, Georges

    2008-02-01

    Although nephrectomy by open surgery is the most used technique for the extraction of kidney transplants in the living donor, nephrectomy under laparaoscopy is increasingly practiced. Laparoscopic nephrectomy is less invasive and performed under videoscopy control, after insufflation of the peritoneal cavity. Three to four incisions are done in order to enter the surgical instruments. The kidney is extracted through a horizontal sus-pubic incision. The exposition is either exclusively transperitoneal, retroperitoneal or hand assisted. The advantages of laparoscopy are esthetical, financial due to a shorter hospitalisation and a quicker recovery, as well a confort for the donor. The disadvantages are a longer warm ischemia time and possibly a higher risk of delayed graft function. Randomised studies having compared laparoscopy and open surgery in the living donor have not find any significant difference regarding the per- and perioperative in the complications. PMID:18160357

  20. Unexpected CEP290 mRNA Splicing in a Humanized Knock-In Mouse Model for Leber Congenital Amaurosis

    PubMed Central

    Garanto, Alejandro; van Beersum, Sylvia E. C.; Peters, Theo A.; Roepman, Ronald; Cremers, Frans P. M.; Collin, Rob W. J.

    2013-01-01

    Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations. PMID:24223178

  1. Regulation of Telomerase Alternative Splicing: A New Target for Chemotherapy

    PubMed Central

    Wong, Mandy S.; Chen, Ling; Foster, Christopher; Kainthla, Radhika; Shay, Jerry W.; Wright, Woodring E.

    2013-01-01

    SUMMARY Telomerase is present in human cancer cells but absent in most somatic tissues. The mRNA of human telomerase (hTERT) is alternatively spliced into mostly non-functional products. We sought to understand splicing so we could decrease functional splice isoforms to reduce telomerase activity to complement direct enzyme inhibition. Unexpectedly, minigenes containing hTERT exons 5–10 flanked by 150–300bp intronic sequences did not produce alternative splicing. A 1.1kb region of 38bp repeats ~2kb from the exon 6/intron junction restored exclusion of exons 7/8. An element within intron 8, also >1kb from intron/exon junctions, modulated this effect. Transducing an oligonucleotide complementary to this second element increased non-functional hTERT mRNA from endogenous telomerase. These results demonstrate the potential of manipulating hTERT splicing for both chemotherapy and regenerative medicine, and provide the first specific sequences deep within introns that regulate alternative splicing in mammalian cells by mechanisms other than introducing cryptic splice sites. PMID:23562158

  2. Method of predicting Splice Sites based on signal interactions

    PubMed Central

    Churbanov, Alexander; Rogozin, Igor B; Deogun, Jitender S; Ali, Hesham

    2006-01-01

    Background Predicting and proper ranking of canonical splice sites (SSs) is a challenging problem in bioinformatics and machine learning communities. Any progress in SSs recognition will lead to better understanding of splicing mechanism. We introduce several new approaches of combining a priori knowledge for improved SS detection. First, we design our new Bayesian SS sensor based on oligonucleotide counting. To further enhance prediction quality, we applied our new de novo motif detection tool MHMMotif to intronic ends and exons. We combine elements found with sensor information using Naive Bayesian Network, as implemented in our new tool SpliceScan. Results According to our tests, the Bayesian sensor outperforms the contemporary Maximum Entropy sensor for 5' SS detection. We report a number of putative Exonic (ESE) and Intronic (ISE) Splicing Enhancers found by MHMMotif tool. T-test statistics on mouse/rat intronic alignments indicates, that detected elements are on average more conserved as compared to other oligos, which supports our assumption of their functional importance. The tool has been shown to outperform the SpliceView, GeneSplicer, NNSplice, Genio and NetUTR tools for the test set of human genes. SpliceScan outperforms all contemporary ab initio gene structural prediction tools on the set of 5' UTR gene fragments. Conclusion Designed methods have many attractive properties, compared to existing approaches. Bayesian sensor, MHMMotif program and SpliceScan tools are freely available on our web site. Reviewers This article was reviewed by Manyuan Long, Arcady Mushegian and Mikhail Gelfand. PMID:16584568

  3. Regulation of alternative splicing of tau exon 10.

    PubMed

    Qian, Wei; Liu, Fei

    2014-04-01

    The neuronal microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in the brains of individuals with Alzheimer's disease and related neurodegenerative disorders. The adult human brain expresses six isoforms of tau generated by alternative splicing of exons 2, 3, and 10 of its pre-mRNA. Exon 10 encodes the second microtubule-binding repeat of tau. Its alternative splicing produces tau isoforms with either three or four microtubule-binding repeats, termed 3R-tau and 4Rtau. In the normal adult human brain, the level of 3R-tau is approximately equal to that of 4R-tau. Several silent and intronic mutations of the tau gene associated with FTDP-17T (frontotemporal dementia with Parkinsonism linked to chromosome 17 and specifically characterized by tau pathology) only disrupt exon 10 splicing, but do not influence the primary sequence of the tau protein. Thus, abnormal exon 10 splicing is sufficient to cause neurodegeneration and dementia. Here, we review the regulation of tau exon 10 splicing by cis-elements and trans-factors and summarize all the mutations associated with FTDP-17T and related tauopathies. The findings suggest that correction of exon 10 splicing may be a potential target for tau exon 10 splicing-related tauopathies. PMID:24627328

  4. Genome-wide survey of Alternative Splicing in Sorghum Bicolor.

    PubMed

    Panahi, Bahman; Abbaszadeh, Bahram; Taghizadeghan, Mehdi; Ebrahimie, Esmaeil

    2014-07-01

    Sorghum bicolor is a member of grass family which is an attractive model plant for genome study due to interesting genome features like low genome size. In this research, we performed comprehensive investigation of Alternative Splicing and ontology aspects of genes those have undergone these events in sorghum bicolor. We used homology based alignments between gene rich transcripts, represented by tentative consensus (TC) transcript sequences, and genomic scaffolds to deduce the structure of genes and identify alternatively spliced transcripts in sorghum. Using homology mapping of assembled expressed sequence tags with genomics data, we identified 2,137 Alternative Splicing events in S. bicolor. Our study showed that complex events and intron retention are the main types of Alternative Splicing events in S. bicolor and highlights the prevalence of splicing site recognition for definition of introns in this plant. Annotations of the alternatively spliced genes revealed that they represent diverse biological process and molecular functions, suggesting a fundamental role for Alternative Splicing in affecting the development and physiology of S. bicolor. PMID:25049459

  5. Interaction of human GTP cyclohydrolase I with its splice variants

    PubMed Central

    Pandya, Maya J.; Golderer, Georg; Werner, Ernst R.; Werner-Felmayer, Gabriele

    2006-01-01

    Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity. PMID:16848765

  6. Evolution of a tissue-specific splicing network

    PubMed Central

    Taliaferro, J. Matthew; Alvarez, Nehemiah; Green, Richard E.; Blanchette, Marco; Rio, Donald C.

    2011-01-01

    Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner. PMID:21406555

  7. Inference of Splicing Regulatory Activities by Sequence Neighborhood Analysis

    PubMed Central

    Stadler, Michael B; Shomron, Noam; Yeo, Gene W; Schneider, Aniket; Xiao, Xinshu; Burge, Christopher B

    2006-01-01

    Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences. PMID:17121466

  8. The Silent Sway of Splicing by Synonymous Substitutions.

    PubMed

    Mueller, William F; Larsen, Liza S Z; Garibaldi, Angela; Hatfield, G Wesley; Hertel, Klemens J

    2015-11-13

    Alternative splicing diversifies mRNA transcripts in human cells. This sequence-driven process can be influenced greatly by mutations, even those that do not change the protein coding potential of the transcript. Synonymous mutations have been shown to alter gene expression through modulation of splicing, mRNA stability, and translation. Using a synonymous position mutation library in SMN1 exon 7, we show that 23% of synonymous mutations across the exon decrease exon inclusion, suggesting that nucleotide identity across the entire exon has been evolutionarily optimized to support a particular exon inclusion level. Although phylogenetic conservation scores are insufficient to identify synonymous positions important for exon inclusion, an alignment of organisms filtered based on similar exon/intron architecture is highly successful. Although many of the splicing neutral mutations are observed to occur, none of the exon inclusion reducing mutants was found in the filtered alignment. Using the modified phylogenetic comparison as an approach to evaluate the impact on pre-mRNA splicing suggests that up to 45% of synonymous SNPs are likely to alter pre-mRNA splicing. These results demonstrate that coding and pre-mRNA splicing pressures co-evolve and that a modified phylogenetic comparison based on the exon/intron architecture is a useful tool in identifying splice altering SNPs. PMID:26424794

  9. In vitro splicing of fibronectin pre-mRNAs.

    PubMed Central

    Norton, P A; Hynes, R O

    1990-01-01

    We have investigated the alternative splicing of the EIIIB exon of the rat fibronectin gene. Mini-gene constructs containing this exon and portions of adjacent introns and exons, when transfected into HeLa cells, are transcribed and spliced, but omit the EIIIB exon. In vitro, HeLa nuclear extracts similarly splice out (skip) the EIIIB exon from similarly structured transcripts. Therefore, the HeLa splicing apparatus recognizes as atypical the EIIIB exon and its flanking intron sequences, both in vivo and in vitro. We also report that alterations in the ionic conditions of the in vitro splicing reaction can promote the initiation of EIIIB exon inclusion, as reflected by the formation of intermediate and product RNAs related to the removal of the intron upstream of EIIIB. Processing of this intron correlates with the formation of complexes resembling intermediates in spliceosome assembly. The branch sites involved in this alternative processing pathway are rather distant from the EIIIB 3' splice site, and lie within a region which is well conserved in the fibronectin genes of other species. Thus, the intron upstream of EIIIB shows singular structure and behavior which probably have a bearing on the regulated alternative splicing of this exon. Images PMID:2377454

  10. A 5' splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated pro alpha 1(I) chains with a non-collagenous insertion destabilizing the triple helix.

    PubMed Central

    Bateman, J F; Chan, D; Moeller, I; Hannagan, M; Cole, W G

    1994-01-01

    A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3' limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7945197

  11. A novel synonymous mutation in the MPZ gene causing an aberrant splicing pattern and Charcot-Marie-Tooth disease type 1b.

    PubMed

    Corrado, L; Magri, S; Bagarotti, A; Carecchio, M; Piscosquito, G; Pareyson, D; Varrasi, C; Vecchio, D; Zonta, A; Cantello, R; Taroni, F; D'Alfonso, S

    2016-08-01

    Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy with a heterogeneous genetic background. Here, we describe two CMT1B families with a mild sensory-motor neuropathy and a novel synonymous variant (c.309G > T, p.G103G) in exon 3 of the MPZ gene. Next generation sequencing analysis on a 94 CMT gene panel showed no mutations in other disease genes. In vitro splicing assay and mRNA expression analysis indicated that the c.309T variant enhances a cryptic donor splice site at position c.304 resulting in the markedly increased expression of the r.304_448del alternative transcript in patients' cells. This transcript is predicted to encode a truncated P0 protein (p.V102Cfs11*) lacking the transmembrane domain, thus suggesting a possible haploinsufficiency mechanism for this mutation. This is the third reported synonymous MPZ variant associated with CMT1 and affecting splicing. These data confirm the functional impact of synonymous variants on MPZ splicing and their possible role as disease-causing mutations rather than silent polymorphisms. PMID:27344971

  12. 30 CFR 75.810 - High-voltage trailing cables; splices.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false High-voltage trailing cables; splices. 75.810... § 75.810 High-voltage trailing cables; splices. In the case of high-voltage cables used as trailing cables, temporary splices shall not be used and all permanent splices shall be made in accordance...

  13. 30 CFR 75.810 - High-voltage trailing cables; splices.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false High-voltage trailing cables; splices. 75.810... § 75.810 High-voltage trailing cables; splices. In the case of high-voltage cables used as trailing cables, temporary splices shall not be used and all permanent splices shall be made in accordance...

  14. 30 CFR 75.810 - High-voltage trailing cables; splices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false High-voltage trailing cables; splices. 75.810... § 75.810 High-voltage trailing cables; splices. In the case of high-voltage cables used as trailing cables, temporary splices shall not be used and all permanent splices shall be made in accordance...

  15. 30 CFR 75.810 - High-voltage trailing cables; splices.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false High-voltage trailing cables; splices. 75.810... § 75.810 High-voltage trailing cables; splices. In the case of high-voltage cables used as trailing cables, temporary splices shall not be used and all permanent splices shall be made in accordance...

  16. Influence of Intron Length on Alternative Splicing of CD44

    PubMed Central

    Bell, Martyn V.; Cowper, Alison E.; Lefranc, Marie-Paule; Bell, John I.; Screaton, Gavin R.

    1998-01-01

    Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained. PMID:9742110

  17. Utilization of Deceased Donor Kidneys to Initiate Living Donor Chains.

    PubMed

    Melcher, M L; Roberts, J P; Leichtman, A B; Roth, A E; Rees, M A

    2016-05-01

    We propose that some deceased donor (DD) kidneys be allocated to initiate nonsimultaneous extended altruistic donor chains of living donor (LD) kidney transplants to address, in part, the huge disparity between patients on the DD kidney waitlist and available donors. The use of DD kidneys for this purpose would benefit waitlisted candidates in that most patients enrolled in kidney paired donation (KPD) systems are also waitlisted for a DD kidney transplant, and receiving a kidney through the mechanism of KPD will decrease pressure on the DD pool. In addition, a LD kidney usually provides survival potential equal or superior to that of DD kidneys. If KPD chains that are initiated by a DD can end in a donation of an LD kidney to a candidate on the DD waitlist, the quality of the kidney allocated to a waitlisted patient is likely to be improved. We hypothesize that a pilot program would show a positive impact on patients of all ethnicities and blood types. PMID:26833680

  18. 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. PMID:17374133

  19. Gene and splicing therapies for neuromuscular diseases.

    PubMed

    Benchaouir, Rachid; Robin, Valerie; Goyenvalle, Aurelie

    2015-01-01

    Neuromuscular disorders (NMD) are heterogeneous group of genetic diseases characterized by muscle weakness and wasting. Duchenne Muscular dystrophy (DMD) and Spinal muscular atrophy (SMA) are two of the most common and severe forms in humans and although the molecular mechanisms of these diseases have been extensively investigated, there is currently no effective treatment. However, new gene-based therapies have recently emerged with particular noted advances in using conventional gene replacement strategies and RNA-based technology. Whilst proof of principle have been demonstrated in animal models, several clinical trials have recently been undertaken to investigate the feasibility of these strategies in patients. In particular, antisense mediated exon skipping has shown encouraging results and hold promise for the treatment of dystrophic muscle. In this review, we summarize the recent progress of therapeutic approaches to neuromuscular diseases, with an emphasis on gene therapy and splicing modulation for DMD and SMA, focusing on the advantages offered by these technologies but also their challenges. PMID:25961553

  20. Splicing modulation therapy in the treatment of genetic diseases

    PubMed Central

    Arechavala-Gomeza, Virginia; Khoo, Bernard; Aartsma-Rus, Annemieke

    2014-01-01

    Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. PMID:25506237

  1. Mass fusion splicing machine for ribbon-type optical fibers

    NASA Astrophysics Data System (ADS)

    Osaka, K.; Yanagi, T.; Asano, Y.

    1986-11-01

    A mass fusion splicer was designed and manufactured. Using this splicer, mass fusion splicing of optical fiber ribbons was investigated. Ten-fiber ribbon tapes were cut and spliced at an average loss of 0.08 dB for GI and 0.24 dB for SM. They were reinforced by heat-shrinkable tubes with EVA adhesive improved for ribbon tape. An average tensile strength until break was about 3.2 kg soon after splice and about 8.3 kg after reinforcement.

  2. Genome-wide profiling of alternative splicing in Alzheimer's disease

    PubMed Central

    Lai, Mitchell K.P.; Esiri, Margaret M.; Tan, Michelle G.K.

    2014-01-01

    Alternative splicing is a highly regulated process which generates transcriptome and proteome diversity through the skipping or inclusion of exons within gene loci. Identification of aberrant alternative splicing associated with human diseases has become feasible with the development of new genomic technologies and powerful bioinformatics. We have previously reported genome-wide gene alterations in the neocortex of a well-characterized cohort of Alzheimer's disease (AD) patients and matched elderly controls using a commercial exon microarray platform [1]. Here, we provide detailed description of analyses aimed at identifying differential alternative splicing events associated with AD. PMID:26484111

  3. Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes.

    PubMed

    Simpson, Craig G; Fuller, John; Calixto, Cristiane P G; McNicol, Jim; Booth, Clare; Brown, John W S; Staiger, Dorothee

    2016-01-01

    Posttranscriptional control makes an important contribution to circadian regulation of gene expression. In higher plants, alternative splicing is particularly prevalent upon abiotic and biotic stress and in the circadian system. Here we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) to monitor alternative splicing events. The use of the panel allows the quantification of changes in the proportion of splice isoforms between different samples, e.g., different time points, different tissues, genotypes, ecotypes, or treatments. PMID:26867620

  4. The Resistance and Strength of Soft Solder Splices between Conductors in MICE Coils

    SciTech Connect

    Wu, Hong; Pan, Heng; Green, Michael A; Dietderich, Dan; Gartner, T. E.; Higley, Hugh C; Mentink, M.; Xu, FengYu; Trillaud, F.; Liu, X. K.; Wang, Li; Zheng, S. X.; Tam, D.G.

    2010-08-03

    Two of the three types of MICE magnets will have splices within their coils. The MICE coupling coils may have as many as fifteen one-meter long splices within them. Each of the MICE focusing coils may have a couple of 0.25-meter long conductor splices. Equations for the calculation of resistance of soldered lap splices of various types are presented. This paper presents resistance measurements of soldered lap splices of various lengths. Measured splice resistance is shown for one-meter long splices as a function of the fabrication method. Another important consideration is the strength of the splices. The measured breaking stress of splices of various lengths is presented in this paper. Tin-lead solders and tin-silver solders were used for the splices that were tested. From the data given in this report, the authors recommend that the use of lead free solders be avoided for low temperature coils.

  5. Dietary uptake efficiency of 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl in yellow perch and rainbow trout: Role of dietary and body lipids

    SciTech Connect

    Dabrowska, H.; Fisher, S.W.; Dabrowski, K.; Staubus, A.E.

    1999-05-01

    Dietary uptake efficiency ({alpha}) and eliminate rate constants (k{sub d}) of 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl (HCBP) were determined in two fish species, yellow perch and rainbow trout, to investigate the influence of dietary and body lipid levels on bioaccumulation. Groups of juvenile fish with significant differences in percent body lipid were fed with a low-fat(LF) or high-fat(HF) diet spiked with 5 or 50 ppb of {sup 14}C-HCBP for 32 d. Thereafter, fish were fed an uncontaminated LF or HF diet to allow for elimination of HCBP. Feeding and growth rates were quantified. There were eight fish lipid-dietary lipid-HCBP concentration exposure combinations for each species. Four fish from each exposure were collected at the beginning of the study and at 10--17-d intervals during exposure and elimination periods for lipid and {sup 14}C-HCBP analysis. The {alpha} values ranged from 74 to 95% in yellow perch and from 79 to 99% in rainbow trout. The greatest {alpha} values, of 95 to 99%, were found in fish given diets with 5 ppb HCBP. Uptake of HCBP was influenced by both dietary and body lipids and depended on the current status of both lipid pools. The elimination rate constants were in the range of 0.000 to 0.004 d{sup {minus}1} in yellow perch and 0.003 to 0.010 d{sup {minus}1} in rainbow trout. No significant differences in elimination rate constants between HF and LF fish groups were found. In fish on a constant dietary lipid regime, the k{sub d} values tended to be less in HF than in LF fish. However, in fish groups offered diets with a change in lipid regime. The k{sub d} tended to be greater. Lipid x time interactions in the HF and LF fish groups undergoing a change in lipid regime indicated that the k{sub d} values, like the {alpha} values, were influenced by both lipid pools. Changes in elimination rates due to dietary/body lipid status impacted BAFs more strongly than changes in uptake efficiencies. The BAFs were in the range of 1.11 to 2

  6. Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: Evidence for involvement of splicing regulatory proteins

    PubMed Central

    Huo, Qing; Kayikci, Melis; Odermatt, Philipp; Meyer, Kathrin; Michels, Olivia; Saxena, Smita; Ule, Jernej; Schümperli, Daniel

    2014-01-01

    Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations. PMID:25692239

  7. Hydrogen Peroxide Alters Splicing of Soluble Guanylyl Cyclase and Selectively Modulates Expression of Splicing Regulators in Human Cancer Cells

    PubMed Central

    Cote, Gilbert J.; Zhu, Wen; Thomas, Anthony; Martin, Emil; Murad, Ferid; Sharina, Iraida G.

    2012-01-01

    Background Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER). Methodology/Principal Findings Here we report that splicing of C-α1 sGC can be modulated by H2O2 treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H2O2 treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H2O2 occurs at the protein level with variable regulation observed in different breast cancer cells. Conclusions/Significance Our data demonstrate that H2O2 regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H2O2 treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress. PMID:22911749

  8. Defective control of pre–messenger RNA splicing in human disease

    PubMed Central

    Shkreta, Lulzim

    2016-01-01

    Examples of associations between human disease and defects in pre–messenger RNA splicing/alternative splicing are accumulating. Although many alterations are caused by mutations in splicing signals or regulatory sequence elements, recent studies have noted the disruptive impact of mutated generic spliceosome components and splicing regulatory proteins. This review highlights recent progress in our understanding of how the altered splicing function of RNA-binding proteins contributes to myelodysplastic syndromes, cancer, and neuropathologies. PMID:26728853

  9. Documented deaths of hepatic lobe donors for living donor liver transplantation.

    PubMed

    Trotter, James F; Adam, Rene; Lo, Chung Mau; Kenison, Jeremy

    2006-10-01

    The actual risk of death in hepatic lobe donors for living donor liver transplantation (LDLT) is unknown because of the lack of a comprehensive database. In the absence of a definitive estimate of the risk of donor death, the medical literature has become replete with anecdotal reports of donor deaths, many of which cannot be substantiated. Because donor death is one of the most important outcomes of LDLT, we performed a comprehensive survey of the medical and lay literature to provide a referenced source of worldwide donor deaths. We reviewed all published articles from the medical literature on LDLT and searched the lay literature for donor deaths from 1989 to February 2006. We classified each death as "definitely," "possibly," or "unlikely" related to donor surgery. We identified 19 donor deaths (and one additional donor in a chronic vegetative state). Thirteen deaths and the vegetative donor were "definitely," 2 were "possibly," and 4 were "unlikely" related to donor surgery. The estimated rate of donor death "definitely" related to donor surgery is 0.15%. The rate of donor death which is "definitely" or "possibly" related to the donor surgery is 0.20%. This analysis provides a source document of all identifiable living liver donor deaths, provides a better estimate of donor death rate, and may provide an impetus for centers with unreported deaths to submit these outcomes to the liver transplantation community. PMID:16952175

  10. Single-Donor Leukophoretic Technique

    NASA Technical Reports Server (NTRS)

    Eberhardt, R. N.

    1977-01-01

    Leukocyte separation-and-retrieval device utilizes granulocyte and monocyte property of leukoadhesion to glass surfaces as basis of their separation from whole blood. Device is used with single donor technique and has application in biological and chemical processing, veterinary research and clinical care.

  11. Physician Migration: Donor Country Impact

    ERIC Educational Resources Information Center

    Aluwihare, A. P. R.

    2005-01-01

    Physician migration from the developing to developed region of a country or the world occurs for reasons of financial, social, and job satisfaction. It is an old phenomenon that produces many disadvantages for the donor region or nation. The difficulties include inequities with the provision of health services, financial loss, loss of educated…

  12. Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana

    PubMed Central

    Raczynska, Katarzyna Dorota; Simpson, Craig G.; Ciesiolka, Adam; Szewc, Lukasz; Lewandowska, Dominika; McNicol, Jim; Szweykowska-Kulinska, Zofia; Brown, John W. S.; Jarmolowski, Artur

    2010-01-01

    The nuclear cap-binding protein complex (CBC) participates in 5′ splice site selection of introns that are proximal to the mRNA cap. However, it is not known whether CBC has a role in alternative splicing. Using an RT–PCR alternative splicing panel, we analysed 435 alternative splicing events in Arabidopsis thaliana genes, encoding mainly transcription factors, splicing factors and stress-related proteins. Splicing profiles were determined in wild type plants, the cbp20 and cbp80(abh1) single mutants and the cbp20/80 double mutant. The alternative splicing events included alternative 5′ and 3′ splice site selection, exon skipping and intron retention. Significant changes in the ratios of alternative splicing isoforms were found in 101 genes. Of these, 41% were common to all three CBC mutants and 15% were observed only in the double mutant. The cbp80(abh1) and cbp20/80 mutants had many more changes in alternative splicing in common than did cbp20 and cbp20/80 suggesting that CBP80 plays a more significant role in alternative splicing than CBP20, probably being a platform for interactions with other splicing factors. Cap-binding proteins and the CBC are therefore directly involved in alternative splicing of some Arabidopsis genes and in most cases influenced alternative splicing of the first intron, particularly at the 5′ splice site. PMID:19864257

  13. Splicing Regulators and Their Roles in Cancer Biology and Therapy

    PubMed Central

    da Silva, Maria Roméria; Moreira, Gabriela Alves; Gonçalves da Silva, Ronni Anderson; de Almeida Alves Barbosa, Éverton; Pais Siqueira, Raoni; Teixera, Róbson Ricardo; Almeida, Márcia Rogéria; Silva Júnior, Abelardo; Fietto, Juliana Lopes Rangel; Bressan, Gustavo Costa

    2015-01-01

    Alternative splicing allows cells to expand the encoding potential of their genomes. In this elegant mechanism, a single gene can yield protein isoforms with even antagonistic functions depending on the cellular physiological context. Alterations in splicing regulatory factors activity in cancer cells, however, can generate an abnormal protein expression pattern that promotes growth, survival, and other processes, which are relevant to tumor biology. In this review, we discuss dysregulated alternative splicing events and regulatory factors that impact pathways related to cancer. The SR proteins and their regulatory kinases SRPKs and CLKs have been frequently found altered in tumors and are examined in more detail. Finally, perspectives that support splicing machinery as target for the development of novel anticancer therapies are discussed. PMID:26273588

  14. Designing oligo libraries taking alternative splicing into account

    NASA Astrophysics Data System (ADS)

    Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

    2001-06-01

    We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

  15. 3. Detail of beam splice and column capital on the ...

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

    3. Detail of beam splice and column capital on the second floor of the Cloth Room Building/Old Bleach House, Monadnock Mills. Beam and column edges are chamfered. - Monadnock Mills, 15 Water Street, Claremont, Sullivan County, NH

  16. Chord Splicing & Joining Detail; Chord & CrossBracing Joint Details; ...

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

    Chord Splicing & Joining Detail; Chord & Cross-Bracing Joint Details; Cross Bracing Center Joint Detail; Chord & Diagonal Joint Detail - Vermont Covered Bridge, Highland Park, spanning Kokomo Creek at West end of Deffenbaugh Street (moved to), Kokomo, Howard County, IN

  17. Splicing: is there an alternative contribution to Parkinson's disease?

    PubMed

    La Cognata, Valentina; D'Agata, Velia; Cavalcanti, Francesca; Cavallaro, Sebastiano

    2015-10-01

    Alternative splicing is a crucial mechanism of gene expression regulation that enormously increases the coding potential of our genome and represents an intermediate step between messenger RNA (mRNA) transcription and protein posttranslational modifications. Alternative splicing occupies a central position in the development and functions of the nervous system. Therefore, its deregulation frequently leads to several neurological human disorders. In the present review, we provide an updated overview on the impact of alternative splicing in Parkinson's disease (PD), the second most common neurodegenerative disorder worldwide. We will describe the alternative splicing of major PD-linked genes by collecting the current evidences about this intricate and not carefully explored aspect. Assessing the role of this mechanism on PD pathobiology may represent a central step toward an improved understanding of this complex disease. PMID:25980689

  18. Network of evolutionary processors with splicing rules and permitting context.

    PubMed

    Choudhary, Ashish; Krithivasan, Kamala

    2007-02-01

    In this paper we consider networks of evolutionary processors with splicing rules and permitting context (NEPPS) as language generating and computational devices. Such a network consists of several processors placed on the nodes of a virtual graph and are able to perform splicing (which is a biologically motivated operation) on the words present in that node, according to the splicing rules present there. Before applying the splicing operation on words, we check for the presence of certain symbols (permitting context) in the strings on which the rule is applied. Each node is associated with an input and output filter. When the filters are based on random context conditions, one gets the computational power of Turing machines with networks of size two. We also show how these networks can be used to solve NP-complete problems in linear time. PMID:17045388

  19. Determinants of Plant U12-Dependent Intron Splicing Efficiency

    PubMed Central

    Lewandowska, Dominika; Simpson, Craig G.; Clark, Gillian P.; Jennings, Nikki S.; Barciszewska-Pacak, Maria; Lin, Chiao-Feng; Makalowski, Wojciech; Brown, John W.S.; Jarmolowski, Artur

    2004-01-01

    Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated. PMID:15100401

  20. Characterization of the Wilson disease gene: Genomic organization; alternative splicing; structure/function predictions; and population frequencies of disease-specific mutations

    SciTech Connect

    Petrukhin, K.; Chernov, I.; Ross, B.M.

    1994-09-01

    The Wilson disease (WD) gene has recently been identified as a putative copper-transporting ATPase with high amino acid similarity with the Menkes disease (MNK) gene. We have further characterized the WD gene by extending the 5{prime}-coding and non-coding DNA sequence and elucidating the intron/exon structure and genomic organization. Analysis of RNA transcripts from liver, brain, kidney and placenta reveals extensive alternative splicing which may provide a mechanism to regulate the quantity of functional protein product. Comparative sequence analysis shows that WD and MNK belong to the sub-family of heavy metal-transporting ATPases with several characterizing features which include unique amino acid motifs and distinct N-terminal and C-terminal transmembrane structure. Our data indicate that the 600 amino acid metal binding portion of the WD and MNK proteins was formed by gene duplication events and splicing of the 6 metal binding domain segment to a common ancestral protein. We have raised a WD-specific anti-peptide antibody to the N-terminal region and are beginning to explore the cellular and intracellular location of the WD protein. The metal-binding segment of the WD protein has been expressed in E. coli and metal binding assays are underway to characterize this aspect of the protein`s function. We have identified numerous disease-specific mutations and developed a rapid {open_quotes}reverse dot blot{close_quotes} screening protocol to determine mutation frequencies in different populations. The most common mutation disrupts the characteristic SEHP motif and accounts for more than 40% of WD cases in North American, Russian, and Swedish populations. This mutation has not been observed in our limited Sicilian sample.

  1. Tissue-specific alternative splicing of TCF7L2.

    PubMed

    Prokunina-Olsson, Ludmila; Welch, Cullan; Hansson, Ola; Adhikari, Neeta; Scott, Laura J; Usher, Nicolle; Tong, Maurine; Sprau, Andrew; Swift, Amy; Bonnycastle, Lori L; Erdos, Michael R; He, Zhi; Saxena, Richa; Harmon, Brennan; Kotova, Olga; Hoffman, Eric P; Altshuler, David; Groop, Leif; Boehnke, Michael; Collins, Francis S; Hall, Jennifer L

    2009-10-15

    Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r(2) = 0.84-0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164-FJ010174. PMID:19602480

  2. The landscape of alternative splicing in cervical squamous cell carcinoma

    PubMed Central

    Guo, Peng; Wang, Dan; Wu, Jun; Yang, Junjun; Ren, Tong; Zhu, Baoli; Xiang, Yang

    2015-01-01

    Background Alternative splicing (AS) is a key regulatory mechanism in protein synthesis and proteome diversity. In this study, we identified alternative splicing events in four pairs of cervical squamous cell carcinoma (CSCC) and adjacent nontumor tissues using RNA sequencing. Methods The transcripts of the four paired samples were thoroughly analyzed by RNA sequencing. SpliceMap software was used to detect the splicing junctions. Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted to detect the alternative spliced genes-related signal pathways. The alternative spliced genes were validated by reverse transcription-polymerase chain reaction (RT-PCR). Results There were 35 common alternative spliced genes in the four CSCC samples; they were novel and CSCC specific. Sixteen pathways were significantly enriched (P<0.05). One novel 5′AS site in the KLHDC7B gene, encoding kelch domain-containing 7B, and an exon-skipping site in the SYCP2 gene, encoding synaptonemal complex 2, were validated by RT-PCR. The KLHDC7B gene with 5′AS was found in 67.5% (27/40) of CSCC samples and was significantly related with cellular differentiation and tumor size. The exon-skipping site of the SYCP2 gene was found in 35.0% (14/40) of CSCC samples and was significantly related with depth of cervical invasion. Conclusion The KLHDC7B and the SYCP2 genes with alternative spliced events might be involved in the development and progression of CSCC and could be used as biomarkers in the diagnosis and prognosis of CSCC. PMID:25565867

  3. Characterization of the Regulation of CD46 RNA Alternative Splicing.

    PubMed

    Tang, Sze Jing; Luo, Shufang; Ho, Jia Xin Jessie; Ly, Phuong Thao; Goh, Eling; Roca, Xavier

    2016-07-01

    Here we present a detailed analysis of the alternative splicing regulation of human CD46, which generates different isoforms with distinct functions. CD46 is a ubiquitous membrane protein that protects host cells from complement and plays other roles in immunity, autophagy, and cell adhesion. CD46 deficiency causes an autoimmune disorder, and this protein is also involved in pathogen infection and cancer. Before this study, the mechanisms of CD46 alternative splicing remained unexplored even though dysregulation of this process has been associated with autoimmune diseases. We proved that the 5' splice sites of CD46 cassette exons 7 and 8 encoding extracellular domains are defined by noncanonical mechanisms of base pairing to U1 small nuclear RNA. Next we characterized the regulation of CD46 cassette exon 13, whose inclusion or skipping generates different cytoplasmic tails with distinct functions. Using splicing minigenes, we identified multiple exonic and intronic splicing enhancers and silencers that regulate exon 13 inclusion via trans-acting splicing factors like PTBP1 and TIAL1. Interestingly, a common splicing activator such as SRSF1 appears to repress CD46 exon 13 inclusion. We also report that expression of CD46 mRNA isoforms is further regulated by non-sense-mediated mRNA decay and transcription speed. Finally, we successfully manipulated CD46 exon 13 inclusion using antisense oligonucleotides, opening up opportunities for functional studies of the isoforms as well as for therapeutics for autoimmune diseases. This study provides insight into CD46 alternative splicing regulation with implications for its function in the immune system and for genetic disease. PMID:27226545

  4. Enantioselective Intramolecular C-H Insertion of Donor and Donor/Donor Carbenes by a Nondiazo Approach.

    PubMed

    Zhu, Dong; Ma, Jun; Luo, Kui; Fu, Hongguang; Zhang, Li; Zhu, Shifa

    2016-07-11

    The first enantioselective intramolecular C-H insertion and cyclopropanation reactions of donor- and donor/donor-carbenes by a nondiazo approach are reported. The reactions were conducted in a one-pot manner without slow addition and provided the desired dihydroindole, dihydrobenzofuran, tetrahydrofuran, and tetrahydropyrrole derivatives with up to 99 % ee and 100 % atom efficiency. PMID:27265896

  5. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    PubMed Central

    Dlamini, Zodwa; Tshidino, Shonisani C.; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  6. Purifying Selection on Exonic Splice Enhancers in Intronless Genes.

    PubMed

    Savisaar, Rosina; Hurst, Laurence D

    2016-06-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  7. Purifying Selection on Exonic Splice Enhancers in Intronless Genes

    PubMed Central

    Savisaar, Rosina; Hurst, Laurence D.

    2016-01-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  8. Functional studies on the ATM intronic splicing processing element

    PubMed Central

    Lewandowska, Marzena A.; Stuani, Cristiana; Parvizpur, Alireza; Baralle, Francisco E.; Pagani, Franco

    2005-01-01

    In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ∼40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing. PMID:16030351

  9. Tafazzin splice variants and mutations in Barth syndrome.

    PubMed

    Kirwin, Susan M; Manolakos, Athena; Barnett, Sarah Swain; Gonzalez, Iris L

    2014-01-01

    Barth syndrome is caused by mutations in the TAZ (tafazzin) gene on human chromosome Xq28. The human tafazzin gene produces four major mRNA splice variants; two of which have been shown to be functional (TAZ lacking exon 5 and full-length) in complementation studies with yeast and Drosophila. This study characterizes the multiple alternative splice variants of TAZ mRNA and their proportions in blood samples from a cohort of individuals with Barth syndrome (BTHS). Because it has been reported that collection and processing methods can affect the expression of various genes, we tested and chose a stabilizing medium for collecting, shipping and processing of the blood samples of these individuals. In both healthy controls and in BTHS individuals, we found a greater variety of alternatively spliced forms than previously described, with a sizeable proportion of minor splice variants besides the four dominant isoforms. Individuals with certain exonic and intronic splice mutations produce additional mutant mRNAs that could be translated into two or more proteins with different amino acid substitutions in a single individual. A fraction of the minor splice variants is predicted to be non-productive. PMID:24342716

  10. Organization, regulatory sequences, and alternatively spliced transcripts of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) gene

    SciTech Connect

    Sampaio, S.O.; Mei, C.; Butcher, E.C.

    1995-09-01

    The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is expressed selectively at venular sites of lymphocyte extravasation into mucosal lymphoid tissues and lamina propria, where it directs local lymphocyte trafficking. MAdCAM-1 is a multifunctional type I transmembrane adhesion molecule comprising two distal Ig domains involved in {alpha}4{beta}7 integrin binding, a mucin-like region able to display L-selectin-binding carbohydrates, and a membrane-proximal Ig domain homologous to IgA. We show in this work that the MAdCAM-1 gene is located on chromosome 10 and contains five exons. The signal peptide and each one of the three Ig domains are encoded by a distinct exon, whereas the transmembrane, cytoplasmic tail, and 3{prime}-untranslated region of MAdCAM-1 are combined on a single exon. The mucin-like region and the third Ig domain are encoded together on exon 4. An alternatively spliced MAdCAM-1 mRNA is identified that lacks the mucin/IgA-homologous exon 4-encoded sequences. This short variant of MAdCAM-1 may be specialized to support {alpha}4{beta}7-dependent adhesion strengthening, independent of carbohydrate-presenting function. Sequences 5{prime} of the transcription start site include tandem nuclear factor-KB sites; AP-1, AP-2, and signal peptide-1 binding sites; and an estrogen response element. Our findings reinforce the correspondence between the multidomain structure and versatile functions of this vascular addressin, and suggest an additional level of regulation of carbohydrate-presenting capability, and thus of its importance in lectin-mediated vs. {alpha}4{beta}7-dependent adhesive events in lymphocyte trafficking. 46 refs., 6 figs., 1 tab.

  11. Nuclear mutations that block group II RNA splicing in maize chloroplasts reveal several intron classes with distinct requirements for splicing factors.

    PubMed Central

    Jenkins, B D; Kulhanek, D J; Barkan, A

    1997-01-01

    To elucidate mechanisms that regulate chloroplast RNA splicing in multicellular plants, we sought nuclear mutations in maize that result in chloroplast splicing defects. Evidence is presented for two nuclear genes whose function is required for the splicing of group II introns in maize chloroplasts. A mutation in the crs1 (for chloroplast RNA splicing 1) gene blocks the splicing of only the atpF intron, whereas a mutation in the crs2 gene blocks the splicing of many chloroplast introns. In addition, a correlation was observed between the absence of plastid ribosomes and the failure to splice several chloroplast introns. Our results suggest that a chloroplast-encoded factor and a nuclear-encoded factor whose activity requires crs2 function facilitate the splicing of distinct sets of group II introns. These two genetically defined intron sets also differ with regard to intron structure: one set consists of only subgroup IIA introns and the other of only subgroup IIB introns. Therefore, it is likely that distinct splicing factors recognize subgroup-specific features of intron structure or facilitate subgroup-specific aspects of the splicing reaction. Of the 12 pre-mRNA introns in the maize chloroplast genome, only one is normally spliced in both crs2 mutants and in mutants lacking plastid ribosomes, indicating that few, if any, of the group II introns in the chloroplast genome undergo autocatalytic splicing in vivo. PMID:9090875

  12. New biologically active hydrogen sulfide donors.

    PubMed

    Roger, Thomas; Raynaud, Francoise; Bouillaud, Frédéric; Ransy, Céline; Simonet, Serge; Crespo, Christine; Bourguignon, Marie-Pierre; Villeneuve, Nicole; Vilaine, Jean-Paul; Artaud, Isabelle; Galardon, Erwan

    2013-11-25

    Generous donors: The dithioperoxyanhydrides (CH3 COS)2 , (PhCOS)2 , CH3 COSSCO2 Me and PhCOSSCO2 Me act as thiol-activated hydrogen sulfide donors in aqueous buffer solution. The most efficient donor (CH3 COS)2 can induce a biological response in cells, and advantageously replace hydrogen sulfide in ex vivo vascular studies. PMID:24115650

  13. 21 CFR 630.6 - Donor notification.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... GENERAL REQUIREMENTS FOR BLOOD, BLOOD COMPONENTS, AND BLOOD DERIVATIVES § 630.6 Donor notification. (a) Notification of donors. You, an establishment that collects blood or blood components, must make reasonable... of donation of blood or blood components that the donor should not donate in the future; (3)...

  14. Alternative splicing of SV40 early pre-mRNA in vitro.

    PubMed Central

    van Santen, V L; Spritz, R A

    1986-01-01

    Simian virus 40 (SV40) early pre-mRNA is spliced using either of two alternative 5' splice sites and a common 3' splice site to produce two mRNAs that encode the T and t antigens. We have studied alternative splicing of SV40 early pre-mRNA in vitro using a HeLa cell nuclear extract. Synthetic SV40 early transcripts are processed to T and t antigen mRNAs in vitro. As in SV40-infected cells in vivo, cleavage at the T antigen 5' splice site is more efficient than cleavage at the t antigen 5' splice site in vitro, although both of these 5' splice sites are utilized relatively inefficiently in vitro. The ratio of cleavage at the T and t antigen 5' splice sites is not changed significantly by a number of alterations in the conditions under which the in vitro splicing reactions are carried out. Images PMID:3027668

  15. JAK2 Exon 14 Skipping in Patients with Primary Myelofibrosis: A Minor Splice Variant Modulated by the JAK2-V617F Allele Burden

    PubMed Central

    Catarsi, Paolo; Rosti, Vittorio; Morreale, Giacomo; Poletto, Valentina; Villani, Laura; Bertorelli, Roberto; Pedrazzini, Matteo; Zorzetto, Michele; Barosi, Giovanni

    2015-01-01

    Background Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the JAK2 gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of JAK2, lacking exon 14 (JAK2Δ14) was described in patients affected by myeloproliferative diseases. Materials and Methods By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the JAK2 full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors. Results We found that JAK2Δ14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2Δ14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence. Conclusions Increased levels of JAK2 full-length transcript and a small but significant increase in JAK2 exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2Δ14 isoform is related to the pathogenesis of PMF. PMID:25617626

  16. The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration

    NASA Astrophysics Data System (ADS)

    Choudhury, Rajarshi; Roy, Sreerupa Ghose; Tsai, Yihsuan S.; Tripathy, Ashutosh; Graves, Lee M.; Wang, Zefeng

    2014-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA-binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The carboxy-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk (extracellular signal-regulated protein kinase) pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq, we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk-regulated cell proliferation.

  17. Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

    PubMed Central

    Horan, Lucas; Yasuhara, Jiro C.; Kohlstaedt, Lori A.; Rio, Donald C.

    2015-01-01

    Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites. PMID:26545814

  18. Evolutionary Character of Alternative Splicing in Plants

    PubMed Central

    Zhang, Chengjun; Yang, Hong; Yang, Huizhao

    2015-01-01

    Alternative splicing (AS) is one of the most important ways to enhance the functional diversity of genes. Huge amounts of data have been produced by microarray, expressed sequence tag, and RNA-seq, and plenty of methods have been developed specifically for this task. The most frequently asked questions in previous research were as follows. What is the content rate of AS genes among the whole gene set? How many AS types are presented in the genome, and which type is dominant? How about the conservation ability of AS among different species? Which kinds of isoforms from some genes have the environmental response to help individual adaptation? Based on this background, we collected analysis results from 17 species to try to map out the landscape of AS studies in plants. We have noted the shortages of previous results, and we appeal to all scientists working in the AS field to make a standard protocol so that analyses between different projects are comparable. PMID:26819552

  19. Hyperbilirubinemia in normal healthy donors

    PubMed Central

    Arora, Veena; Kulkarni, R. K.; Cherian, Susan; Pillai, Raji; Shivali, M.

    2009-01-01

    The present study was carried out in B.A.R.C. Hospital Blood Bank over a span of five years, and includes 2734 donors. All the bags were screened for HIV, HBsAg, HCV and VDRL and the plasma in the pilot tubes of the blood bags was observed to detect any abnormality in color. In 27 cases plasma was found to be icteric and liver function tests were carried out on these samples. Two donors showed higher SGPT level, and were excluded. No significant increases in liver enzymes were recorded in the others. Causes of icteric plasma in these apparently healthy donors are discussed. Differential diagnosis includes Gilbert’s disease, hemolytic anemia, drug-induced anemia and other hepatic causes of hyperbilirubinemia, of which Gilbert’s disease is most probable cause with a prevalence of 0.91% in our population. As there are no studies to document the safety of the recipients receiving such abnormal colored plasma as well as to document the hazards in its transfusion, the question arises whether to transfuse such units or not. This study highlights this dilemma. A reassessment of existing policies and regulations is merited. PMID:20808649

  20. Changing Pattern of Donor Selection Criteria in Deceased Donor Liver Transplant: A Review of Literature

    PubMed Central

    Routh, Dronacharya; Naidu, Sudeep; Sharma, Sanjay; Ranjan, Priya; Godara, Rajesh

    2013-01-01

    During the last couple of decades, with standardization and progress in surgical techniques, immunosuppression and post liver transplantation patient care, the outcome of liver transplantation has been optimized. However, the principal limitation of transplantation remains access to an allograft. The number of patients who could derive benefit from liver transplantation markedly exceeds the number of available deceased donors. The large gap between the growing list of patients waiting for liver transplantation and the scarcity of donor organs has fueled efforts to maximize existing donor pool and identify new avenues. This article reviews the changing pattern of donor for liver transplantation using grafts from extended criteria donors (elderly donors, steatotic donors, donors with malignancies, donors with viral hepatitis), donation after cardiac death, use of partial grafts (split liver grafts) and other suboptimal donors (hypernatremia, infections, hypotension and inotropic support). PMID:25755521

  1. scaRNAs regulate splicing and vertebrate heart development.

    PubMed

    Patil, Prakash; Kibiryeva, Nataliya; Uechi, Tamayo; Marshall, Jennifer; O'Brien, James E; Artman, Michael; Kenmochi, Naoya; Bittel, Douglas C

    2015-08-01

    Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations. PMID:25916634

  2. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing. PMID:26553470

  3. A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

    PubMed Central

    Skandalis, Adonis; Uribe, Elke

    2004-01-01

    Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase β (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40–60%), whereas the frequency of HPRT splice variants is considerably lower (∼1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed. PMID:15601998

  4. Convergent origins and rapid evolution of spliced leader trans-splicing in Metazoa: Insights from the Ctenophora and Hydrozoa

    PubMed Central

    Derelle, Romain; Momose, Tsuyoshi; Manuel, Michael; Da Silva, Corinne; Wincker, Patrick; Houliston, Evelyn

    2010-01-01

    Replacement of mRNA 5′ UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3′ of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare. PMID:20142326

  5. Analysis of a Splice Array Experiment Elucidates Roles of Chromatin Elongation Factor Spt4–5 in Splicing

    PubMed Central

    2005-01-01

    Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4–5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4–5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4–Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function. PMID:16172632

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

    PubMed Central

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

    2015-01-01

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

  7. TCGASpliceSeq a compendium of alternative mRNA splicing in cancer.

    PubMed

    Ryan, Michael; Wong, Wing Chung; Brown, Robert; Akbani, Rehan; Su, Xiaoping; Broom, Bradley; Melott, James; Weinstein, John

    2016-01-01

    TCGA's RNASeq data represent one of the largest collections of cancer transcriptomes ever assembled. RNASeq technology, combined with computational tools like our SpliceSeq package, provides a comprehensive, detailed view of alternative mRNA splicing. Aberrant splicing patterns in cancers have been implicated in such processes as carcinogenesis, de-differentiation and metastasis. TCGA SpliceSeq (http://bioinformatics.mdanderson.org/TCGASpliceSeq) is a web-based resource that provides a quick, user-friendly, highly visual interface for exploring the alternative splicing patterns of TCGA tumors. Percent Spliced In (PSI) values for splice events on samples from 33 different tumor types, including available adjacent normal samples, have been loaded into TCGA SpliceSeq. Investigators can interrogate genes of interest, search for the genes that show the strongest variation between or among selected tumor types, or explore splicing pattern changes between tumor and adjacent normal samples. The interface presents intuitive graphical representations of splicing patterns, read counts and various statistical summaries, including percent spliced in. Splicing data can also be downloaded for inclusion in integrative analyses. TCGA SpliceSeq is freely available for academic, government or commercial use. PMID:26602693

  8. SR proteins are required for nematode trans-splicing in vitro.

    PubMed Central

    Sanford, J R; Bruzik, J P

    1999-01-01

    SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts. PMID:10411135

  9. Species-specific signals for the splicing of a short Drosophila intron in vitro.

    PubMed Central

    Guo, M; Lo, P C; Mount, S M

    1993-01-01

    The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species. Images PMID:8423778

  10. Sec16 alternative splicing dynamically controls COPII transport efficiency.

    PubMed

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  11. Long-time dynamics through parallel trajectory splicing

    SciTech Connect

    Perez, Danny; Cubuk, Ekin D.; Waterland, Amos; Kaxiras, Efthimios; Voter, Arthur F.

    2015-11-24

    Simulating the atomistic evolution of materials over long time scales is a longstanding challenge, especially for complex systems where the distribution of barrier heights is very heterogeneous. Such systems are difficult to investigate using conventional long-time scale techniques, and the fact that they tend to remain trapped in small regions of configuration space for extended periods of time strongly limits the physical insights gained from short simulations. We introduce a novel simulation technique, Parallel Trajectory Splicing (ParSplice), that aims at addressing this problem through the timewise parallelization of long trajectories. The computational efficiency of ParSplice stems from a speculation strategy whereby predictions of the future evolution of the system are leveraged to increase the amount of work that can be concurrently performed at any one time, hence improving the scalability of the method. ParSplice is also able to accurately account for, and potentially reuse, a substantial fraction of the computational work invested in the simulation. We validate the method on a simple Ag surface system and demonstrate substantial increases in efficiency compared to previous methods. As a result, we then demonstrate the power of ParSplice through the study of topology changes in Ag42Cu13 core–shell nanoparticles.

  12. Sec16 alternative splicing dynamically controls COPII transport efficiency

    PubMed Central

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  13. BRR2a Affects Flowering Time via FLC Splicing

    PubMed Central

    Mahrez, Walid; Shin, Juhyun; Exner, Vivien; Siretskiy, Alexey; Köhler, Claudia

    2016-01-01

    Several pathways control time to flowering in Arabidopsis thaliana through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of BRR2a. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably FLC. A missense mutation of threonine 895 in BRR2a caused defects in FLC splicing and greatly reduced FLC transcript levels. Reduced FLC expression increased transcription of FT and SOC1 leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the brr2a mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that FLC is most sensitive to splicing defects. PMID:27100965

  14. Capturing splicing complexes to study structure and mechanism.

    PubMed

    Jurica, Melissa S; Moore, Melissa J

    2002-11-01

    At its most basic level, pre-mRNA splicing can be described as two coordinated nuclease reactions that cleave an intron at either end and result in ligation of the flanking exons. The fact that these reactions are catalyzed by a approximately 3-MDa behemoth of protein and RNA (the spliceosome) challenges most biochemical and structural approaches currently used to characterize lesser-sized enzymes. In addition to this molecular complexity, the highly dynamic nature of splicing complexes provides additional hurdles for mechanistic studies or three-dimensional structure determination. Thus, the methods used to study the spliceosome often probe individual properties of the machine, but no complete, high-resolution picture of splicing catalysis has yet emerged. To facilitate biochemical and structural studies of native splicing complexes, we recently described purification of the catalytic form of the spliceosome (known as C complex). This native complex is suitable for electron microscopic structure determination by single-particle methods. In this paper, we describe the purification in detail and discuss additional methods for trapping and analyzing other splicing complexes. PMID:12431437

  15. Long-time dynamics through parallel trajectory splicing

    DOE PAGESBeta

    Perez, Danny; Cubuk, Ekin D.; Waterland, Amos; Kaxiras, Efthimios; Voter, Arthur F.

    2015-11-24

    Simulating the atomistic evolution of materials over long time scales is a longstanding challenge, especially for complex systems where the distribution of barrier heights is very heterogeneous. Such systems are difficult to investigate using conventional long-time scale techniques, and the fact that they tend to remain trapped in small regions of configuration space for extended periods of time strongly limits the physical insights gained from short simulations. We introduce a novel simulation technique, Parallel Trajectory Splicing (ParSplice), that aims at addressing this problem through the timewise parallelization of long trajectories. The computational efficiency of ParSplice stems from a speculation strategymore » whereby predictions of the future evolution of the system are leveraged to increase the amount of work that can be concurrently performed at any one time, hence improving the scalability of the method. ParSplice is also able to accurately account for, and potentially reuse, a substantial fraction of the computational work invested in the simulation. We validate the method on a simple Ag surface system and demonstrate substantial increases in efficiency compared to previous methods. As a result, we then demonstrate the power of ParSplice through the study of topology changes in Ag42Cu13 core–shell nanoparticles.« less

  16. BRR2a Affects Flowering Time via FLC Splicing.

    PubMed

    Mahrez, Walid; Shin, Juhyun; Muñoz-Viana, Rafael; Figueiredo, Duarte D; Trejo-Arellano, Minerva S; Exner, Vivien; Siretskiy, Alexey; Gruissem, Wilhelm; Köhler, Claudia; Hennig, Lars

    2016-04-01

    Several pathways control time to flowering in Arabidopsis thaliana through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of BRR2a. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably FLC. A missense mutation of threonine 895 in BRR2a caused defects in FLC splicing and greatly reduced FLC transcript levels. Reduced FLC expression increased transcription of FT and SOC1 leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the brr2a mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that FLC is most sensitive to splicing defects. PMID:27100965

  17. Oncogenes and RNA splicing of human tumor viruses.

    PubMed

    Ajiro, Masahiko; Zheng, Zhi-Ming

    2014-09-01

    Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis. PMID:26038756

  18. Donor free radical explosive composition

    DOEpatents

    Walker, Franklin E. [15 Way Points Rd., Danville, CA 94526; Wasley, Richard J. [4290 Colgate Way, Livermore, CA 94550

    1980-04-01

    An improved explosive composition is disclosed and comprises a major portion of an explosive having a detonation velocity between about 1500 and 10,000 meters per second and a minor amount of a donor additive comprising an organic compound or mixture of organic compounds capable of releasing low molecular weight free radicals or ions under mechanical or electrical shock conditions and which is not an explosive, or an inorganic compound or mixture of inorganic compounds capable of releasing low molecular weight free radicals or ions under mechanical or electrical shock conditions and selected from ammonium or alkali metal persulfates.

  19. Protein kinase C delta (PKCδ) splice variant modulates senescence via hTERT in adipose-derived stem cells

    PubMed Central

    Carter, Gay; Patel, Rekha; Apostolatos, André; Murr, Michel; Cooper, Denise R.

    2014-01-01

    Background Adipose-derived stem cells (ADSC) were isolated and characterized from lean and obese subjects. We previously reported that distinct differences were observed in differentiating lean and obese preadipocytes. Protein kinase C delta (PKCδ) is alternatively spliced and has important roles in apoptosis. PKCδI promotes apoptosis and PKCδVIII promotes survival. Our previous data indicated an increase in the survival kinase, PKCδVIII in ADSC derived from an obese donor. We also determined that obese adipocytes were resistant to apoptosis. Here, we determine the relationship between a survival kinase PKCδVIII and hTERT expression in adipose derived stem cells from a lean and obese subject. Methods We evaluated the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in lean and obese ADSC. The lean and obese ADSC were purchased as cryopreserved cells from ZenBio™ (Research Triangle Park, NC, USA). Analyses were performed using PRISM™ software and analyzed using two-tailed Student’s t-test. Results We observed an increase in telomerase in differentiating obese ADSC using western blot analysis. We determined the levels of hTERT splice variants. hTERT α+/β+ splice variant was increased after transfected of PKCδVIII. We next determined whether PKCδVIII over-expression affected the levels of telomerase. The results indicate an increase in telomerase with PKCδVIII over-expression. Conclusions Over-expression of PKCδVIII in lean ADSC substantially increased expression of hTERT and telomerase. The decreased senescence seen in obese ADSC may in part be attributed to PKCδVIII. Obese ADSC undergo lower senescence and may have increased growth potential. These results propose a larger epigenetic modification in obese ADSC compared to lean ADSC.

  20. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    DOE PAGESBeta

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; et al

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

  1. Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites.

    PubMed

    Semlow, Daniel R; Blanco, Mario R; Walter, Nils G; Staley, Jonathan P

    2016-02-25

    During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3' splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3' to 5' translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome. PMID:26919433

  2. Integrative Analysis of Many RNA-Seq Datasets to Study Alternative Splicing

    PubMed Central

    Li, Wenyuan; Dai, Chao; Kang, Shuli; Zhou, Xianghong Jasmine

    2014-01-01

    Alternative splicing is an important gene regulatory mechanism that dramatically increases the complexity of the proteome. However, how alternative splicing is regulated and how transcription and splicing are coordinated are still poorly understood, and functions of transcript isoforms have been studied only in a few limited cases. Nowadays, RNA-seq technology provides an exceptional opportunity to study alternative splicing on genome-wide scales and in an unbiased manner. With the rapid accumulation of data in public repositories, new challenges arise from the urgent need to effectively integrate many different RNA-seq datasets for study alterative splicing. This paper discusses a set of advanced computational methods that can integrate and analyze many RNA-seq datasets to systematically identify splicing modules, unravel the coupling of transcription and splicing, and predict the functions of splicing isoforms on a genome-wide scale. PMID:24583115

  3. Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein

    SciTech Connect

    Lewin, A.S.; Thomas, J. Jr.; Tirupati, H.K.

    1995-12-01

    This report investigates the coupling between transcription and splicing of a mitochondrial group I intron in Saccharomyces cerevisiae and the effect of the Cbp2 protein on splicing. 65 refs., 7 figs.

  4. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  5. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    SciTech Connect

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

  6. Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3’ Splice Site Recognition

    PubMed Central

    Smith, Lindsay D.; Lucas, Christian M.; Eperon, Ian C.

    2013-01-01

    The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3’ splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5’ of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo. PMID:24155930

  7. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing

    PubMed Central

    Guo, Wei; Schafer, Sebastian; Greaser, Marion L.; Radke, Michael H.; Liss, Martin; Govindarajan, Thirupugal; Maatz, Henrike; Schulz, Herbert; Li, Shijun; Parrish, Amanda M.; Dauksaite, Vita; Vakeel, Padmanabhan; Klaassen, Sabine; Gerull, Brenda; Thierfelder, Ludwig; Regitz-Zagrosek, Vera; Hacker, Timothy A.; Saupe, Kurt W.; Dec, G. William; Ellinor, Patrick T.; MacRae, Calum A.; Spallek, Bastian; Fischer, Robert; Perrot, Andreas; Özcelik, Cemil; Saar, Kathrin; Hubner, Norbert; Gotthardt, Michael

    2013-01-01

    Alternative splicing plays a major role in the adaptation of cardiac function exemplified by the isoform switch of titin, which adjusts ventricular filling. We previously identified a rat strain deficient in titin splicing. Using genetic mapping, we found a loss-of-function mutation in RBM20 as the underlying cause for the pathological titin isoform expression. Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy. We showed that the phenotype of Rbm20 deficient rats resembles the human pathology. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20 dependent regulation of alternative splicing. Additionally to titin we identified a set of 30 genes with conserved regulation between human and rat. This network is enriched for genes previously linked to cardiomyopathy, ion-homeostasis, and sarcomere biology. Our studies emphasize the importance of posttranscriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure. PMID:22466703

  8. Alternative splicing of the androgen receptor in polycystic ovary syndrome.

    PubMed

    Wang, Fangfang; Pan, Jiexue; Liu, Ye; Meng, Qing; Lv, Pingping; Qu, Fan; Ding, Guo-Lian; Klausen, Christian; Leung, Peter C K; Chan, Hsiao Chang; Yao, Weimiao; Zhou, Cai-Yun; Shi, Biwei; Zhang, Junyu; Sheng, Jianzhong; Huang, Hefeng

    2015-04-14

    Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female subfertility. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. Here, we identify two alternative splice variants (ASVs) of the androgen receptor (AR), insertion and deletion isoforms, in granulosa cells (GCs) in ∼62% of patients with PCOS. AR ASVs are strongly associated with remarkable hyperandrogenism and abnormalities in folliculogenesis, and are absent from all control subjects without PCOS. Alternative splicing dramatically alters genome-wide AR recruitment and androgen-induced expression of genes related to androgen metabolism and folliculogenesis in human GCs. These findings establish alternative splicing of AR in GCs as the major pathogenic mechanism for hyperandrogenism and abnormal folliculogenesis in PCOS. PMID:25825716

  9. Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

    PubMed

    Yang, Xinping; Coulombe-Huntington, Jasmin; Kang, Shuli; Sheynkman, Gloria M; Hao, Tong; Richardson, Aaron; Sun, Song; Yang, Fan; Shen, Yun A; Murray, Ryan R; Spirohn, Kerstin; Begg, Bridget E; Duran-Frigola, Miquel; MacWilliams, Andrew; Pevzner, Samuel J; Zhong, Quan; Trigg, Shelly A; Tam, Stanley; Ghamsari, Lila; Sahni, Nidhi; Yi, Song; Rodriguez, Maria D; Balcha, Dawit; Tan, Guihong; Costanzo, Michael; Andrews, Brenda; Boone, Charles; Zhou, Xianghong J; Salehi-Ashtiani, Kourosh; Charloteaux, Benoit; Chen, Alyce A; Calderwood, Michael A; Aloy, Patrick; Roth, Frederick P; Hill, David E; Iakoucheva, Lilia M; Xia, Yu; Vidal, Marc

    2016-02-11

    While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms"). PMID:26871637

  10. Identifying splicing regulatory elements with de Bruijn graphs.

    PubMed

    Badr, Eman; Heath, Lenwood S

    2014-12-01

    Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation. PMID:25393830

  11. A novel biallelic splice site mutation of TECTA causes moderate to severe hearing impairment in an Algerian family.

    PubMed

    Behlouli, Asma; Bonnet, Crystel; Abdi, Samia; Hasbellaoui, Mokhtar; Boudjenah, Farid; Hardelin, Jean-Pierre; Louha, Malek; Makrelouf, Mohamed; Ammar-Khodja, Fatima; Zenati, Akila; Petit, Christine

    2016-08-01

    Congenital deafness is certainly one of the most common monogenic diseases in humans, but it is also one of the most genetically heterogeneous, which makes molecular diagnosis challenging in most cases. Whole-exome sequencing in two out of three Algerian siblings affected by recessively-inherited, moderate to severe sensorineural deafness allowed us to identify a novel splice donor site mutation (c.5272+1G > A) in the gene encoding α-tectorin, a major component of the cochlear tectorial membrane. The mutation was present at the homozygous state in the three affected siblings, and at the heterozygous state in their unaffected, consanguineous parents. To our knowledge, this is the first reported TECTA mutation leading to the DFNB21 form of hearing impairment among Maghrebian individuals suffering from congenital hearing impairment, which further illustrates the diversity of the genes involved in congenital deafness in the Maghreb. PMID:27368438

  12. Gamete donors' expectations and experiences of contact with their donor offspring

    PubMed Central

    Kirkman, Maggie; Bourne, Kate; Fisher, Jane; Johnson, Louise; Hammarberg, Karin

    2014-01-01

    STUDY QUESTION What are the expectations and experiences of anonymous gamete donors about contact with their donor offspring? SUMMARY ANSWER Rather than consistently wanting to remain distant from their donor offspring, donors' expectations and experiences of contact with donor offspring ranged from none to a close personal relationship. WHAT IS KNOWN ALREADY Donor conception is part of assisted reproduction in many countries, but little is known about its continuing influence on gamete donors' lives. STUDY DESIGN, SIZE, DURATION A qualitative research model appropriate for understanding participants' views was employed; semi-structured interviews were conducted during January–March 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS Before 1998, gamete donors in Victoria, Australia, were subject to evolving legislation that allowed them to remain anonymous or (from 1988) to consent to the release of identifying information. An opportunity to increase knowledge of donors' expectations and experiences of contact with their donor offspring recently arose in Victoria when a recommendation was made to introduce mandatory identification of donors on request from their donor offspring, with retrospective effect. Pre-1998 donors were invited through an advertising campaign to be interviewed about their views, experiences and expectations; 36 sperm donors and 6 egg donors participated. MAIN RESULTS AND THE ROLE OF CHANCE This research is unusual in achieving participation by donors who would not normally identify themselves to researchers or government inquiries. Qualitative thematic analysis revealed that most donors did not characterize themselves as parents of their donor offspring. Donors' expectations and experiences of contact with donor offspring ranged from none to a close personal relationship. LIMITATIONS, REASONS FOR CAUTION It is not possible to establish whether participants were representative of all pre-1998 donors. WIDER IMPLICATIONS OF THE FINDINGS Anonymous

  13. A novel splice site mutation in SMARCAL1 results in aberrant exon definition in a child with schimke immunoosseous dysplasia.

    PubMed

    Carroll, Clinton; Hunley, Tracy E; Guo, Yan; Cortez, David

    2015-10-01

    Schimke Immunoosseous Dysplasia (SIOD) is a rare, autosomal recessive disorder of childhood characterized by spondyloepiphyseal dysplasia, focal segmental glomerulosclerosis and renal failure, T-cell immunodeficiency, and cancer in certain instances. Approximately half of patients with SIOD are reported to have biallelic mutations in SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA translocase that localizes to sites of DNA replication and repairs damaged replication forks. We present a novel mutation (NM_014140.3:c.2070+2insT) that results in defective SMARCAL1 mRNA splicing in a child with SIOD. This mutation, within the donor site of intron 12, results in the skipping of exon 12, which encodes part of a critical hinge region connecting the two lobes of the ATPase domain. This mutation was not recognized as deleterious by diagnostic SMARCAL1 sequencing, but discovered through next generation sequencing and found to result in absent SMARCAL1 expression in patient-derived lymphoblasts. The splicing defect caused by this mutation supports the concept of exon definition. Furthermore, it illustrates the need to broaden the search for SMARCAL1 mutations in patients with SIOD lacking coding sequence variants. PMID:25943327

  14. Hypoxia-Induced Alternative Splicing in Endothelial Cells

    PubMed Central

    Weigand, Julia E.; Boeckel, Jes-Niels; Gellert, Pascal; Dimmeler, Stefanie

    2012-01-01

    Background Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells. Methodology/Principal findings Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O2) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max). Conclusions/Significance For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells

  15. Prp40 and early events in splice site definition.

    PubMed

    Becerra, Soraya; Andrés-León, Eduardo; Prieto-Sánchez, Silvia; Hernández-Munain, Cristina; Suñé, Carlos

    2016-01-01

    The alternative splicing (AS) of precursor messenger RNA (pre-mRNA) is a tightly regulated process through which introns are removed to leave the resulting exons in the mRNA appropriately aligned and ligated. The AS of pre-mRNA is a key mechanism for increasing the complexity of proteins encoded in the genome. In humans, more than 90% of genes undergo AS, underscoring the importance of this process in RNA biogenesis. As such, AS misregulation underlies multiple human diseases. The splicing reaction is catalyzed by the spliceosome, a highly dynamic complex that assembles at or near the intron/exon boundaries and undergoes sequential conformational and compositional changes during splicing. The initial recognition of splice sites defines the exons that are going to be removed, which is a critical step in the highly regulated splicing process. Although the available lines of evidence are increasing, the molecular mechanisms governing AS, including the initial interactions occurring at intron/exon boundaries, and the factors that modulate these critical connections by functioning as a scaffold for active-site RNAs or proteins, remain poorly understood. In this review, we summarize the major hallmarks of the initial steps in the splicing process and the role of auxiliary factors that contribute to the assembly of the spliceosomal complex. We also discuss the role of the essential yeast Prp40 protein and its mammalian homologs in the specificity of this pre-mRNA processing event. In addition, we provide the first exhaustive phylogenetic analysis of the molecular evolution of Prp40 family members. WIREs RNA 2016, 7:17-32. doi: 10.1002/wrna.1312 For further resources related to this article, please visit the WIREs website. PMID:26494226

  16. Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates.

    PubMed Central

    Ogden, R C; Lee, M C; Knapp, G

    1984-01-01

    The primary sequences of all the tRNA precursors which contain intervening sequences and which accumulate in the Saccharomyces cerevisiae rnal mutant are presented. A combination of DNA and RNA sequence analysis has led to elucidation of the primary sequence of four hitherto uncharacterized precursors. The location of the intervening sequence has in all cases been unambiguously determined by analysis of the intermediates in the splicing reaction. Secondary structures based upon the tRNA cloverleaf are shown for all the tRNA precursors and discussed with respect to common recognition by the yeast splicing endonuclease. Images PMID:6096826

  17. A chloroplast retrograde signal regulates nuclear alternative splicing

    PubMed Central

    Petrillo, Ezequiel; Herz, Micaela A. Godoy; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J.; Simpson, Craig; Brown, John W. S.; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R.

    2015-01-01

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

  18. Quality improvement in the care of live liver donors: implementation of the Designated Donor Nurse Program.

    PubMed

    LaPointe Rudow, Dianne; Cabello, Charlotte C; Rivellini, Denise

    2010-12-01

    Publications on living donor liver transplant have focused on the medical aspects of donor selection, postoperative management, surgical procedures, and outcomes, but little attention has been given to the nursing implications for care of live liver donors during their inpatient stay. Donor advocates from various disciplines are involved during the initial education and evaluation, but most care after surgery is delivered by an inpatient medical team and bedside nursing staff who are not as familiar with the donor and concepts related to donor advocacy. In an effort to improve the overall donor experience and provide safe, high-quality care to patients undergoing elective partial hepatectomy, our academic medical center began a quality improvement project focused on improving the inpatient stay. Inpatient nursing standards and policies and procedures were developed to ensure that consistent care is delivered. However, the infrequency of living donor liver transplantation makes it nearly impossible to have all transplant program staff on a nursing unit be "experts" on donor care. Therefore, our center determined that, similar to the Independent Donor Advocacy Team, a transplant program needs live donor champions on the nursing unit to mirror the goals of the team. To that end, we developed the concept of the Designated Donor Nurse to care for and advocate for live liver donors during the inpatient stay and also to serve as a resource to their colleagues. PMID:21265291

  19. The living donor advocate: a team approach to educate, evaluate, and manage donors across the continuum.

    PubMed

    Rudow, Dianne LaPointe

    2009-03-01

    Living donor transplant has developed as a direct result of the critical shortage of deceased donors. Federal regulations require transplant programs to appoint an independent donor advocate to ensure safe evaluation and care of live donors. Ethical and pragmatic issues surround the donor advocate. These issues include the composition of a team versus an individual advocate, who appoints them, and the role that the advocate(s) play in the process. A team approach to donor advocacy is recommended. Common goals of the independent donor advocacy team should be protocol development, education, medical and psychosocial evaluation, advocacy, support, and documentation throughout the donation process. The team's involvement should not end with consent and donation but should continue through short- and long-term follow-up and management. Ultimately it is the goal of the independent donor advocacy team to assist donors to advocate for themselves. Once deemed medically and psychologically suitable, donors must determine for themselves what they wish to do and must be free to vocalize this to their team. The decision to donate or not affects the donor first. Optimal outcomes begin with prepared, educated, uncoerced, and motivated donors, and it is the team's goal to help donors reach this point. PMID:19341065

  20. LDLR gene synonymous mutation c.1813C>T results in mRNA splicing variation in a kindred with familial hypercholesterolaemia.

    PubMed

    Ho, Clement K M; Musa, Fathel Rahman; Bell, Christine; Walker, Simon W

    2015-11-01

    Familial hypercholesterolaemia, one of the most common inherited diseases in the general population, is associated with mutations in at least three different genes including the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and protein convertase subtilisin/kexin type 9 (PCSK9) genes. In this report, we describe an unclassified DNA variant (c.1813C>T; p.Leu605Leu) within exon 12 of the LDLR gene in a kindred in which familial hypercholesterolaemia is associated with c.1813C>T heterozygosity. In silico analysis suggested that c.1813C>T might affect splicing of the LDLR gene by creating a cryptic donor splice site, which was confirmed by RT-PCR coupled with cDNA sequencing, to result in the loss of 34 base pairs in the coding sequence. The latter truncated mRNA is predicted to generate a frameshift leading to a premature stop at codon 652 and early termination of the low density lipoprotein receptor polypeptide chain, and thus provides a molecular basis for the hypercholesterolaemic phenotype. This case report highlights the emerging utility of RNA studies for the molecular diagnosis of familial hypercholesterolaemia in patients with potential mRNA splicing variants. PMID:25624525

  1. Management of the inpatient canine blood donor.

    PubMed

    Hohenhaus, A E

    1992-12-01

    The availability of inpatient blood donors as a source for transfusion allows flexibility that is lacking in an outpatient program. Choosing the appropriate dog as a donor is essential to the success of a hospital blood bank. Once a dog becomes a blood donor, routine physical and clinicopathologic examinations are necessary to monitor the animal's health and to ensure the quality of blood products. PMID:1472767

  2. Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila

    PubMed Central

    Duff, Michael O.; Olson, Sara; Wei, Xintao; Garrett, Sandra C.; Osman, Ahmad; Bolisetty, Mohan; Plocik, Alex; Celniker, Susan; Graveley, Brenton R.

    2015-01-01

    Recursive splicing is a process in which large introns are removed in multiple steps by resplicing at ratchet points - 5′ splice sites recreated after splicing1. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene1 and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing2,3. Here, we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues, and cultured cells. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, that depletion of U2AF inhibits recursive splicing, and that the sequence and function of ratchet points are evolutionarily conserved in Drosophila. Finally, we identified four recursively spliced human genes, one of which is also recursively spliced in Drosophila. Together these results indicate that recursive splicing is commonly used in Drosophila, occurs in human and provides insight into the mechanisms by which some large introns are removed. PMID:25970244

  3. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    PubMed

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  4. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    PubMed Central

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  5. Metal boot permits fabrication of hermetically sealed splices in metal sheathed instrumentation cables

    NASA Technical Reports Server (NTRS)

    Chambers, G.

    1966-01-01

    Metal boot splices hard sheathed instrumentation cables used with high temperature strain gages and thermocouples. Silver brazing the conductors together, hermetically seals the splice. This boot is a highly reliable sealed splice which is equally effective at cryogenic temperatures, high temperatures, nuclear environments, and combinations of the above.

  6. The human peripheral benzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

    SciTech Connect

    Lin, D.; Miller, W.L. ); Chang, Y.J.; Strauss, J.F. III )

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondia to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, the authors have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5[prime] untranslated segment. The 5[prime] flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of [open quotes]housekeeping[close quotes] genes. The encoded PBR mRNA is alternately spliced into two forms: [open quotes]authentic[close quotes] PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression of PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [[sup 3]H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease. 36 refs., 6 figs.

  7. Living-donor liver transplantation: current perspective.

    PubMed

    Lobritto, Steven; Kato, Tomoaki; Emond, Jean

    2012-11-01

    The disparity between the number of available deceased liver donors and the number of patients awaiting transplantation continues to be an ongoing issue predisposing to death on the liver transplant waiting list. Deceased donor shortage strategies including the use of extended donor-criteria deceased donor grafts, split liver transplants, and organs harvested after cardiac death have fallen short of organ demand. Efforts to raise donor awareness are ongoing, but the course has been arduous to date. Living donor transplantation is a means to access an unlimited donor organ supply and offers potential advantages to deceased donation. Donor safety remains paramount demanding improvements and innovations in both the donor and recipient operations to ensure superior outcomes. The specialty operation is best preformed at centers with specific expertise and shuttling of select patients to these centers supported by third party payers is critical. Training future surgeons at centers with this specific experience can help disseminate this technology to improve local availability. Ongoing research in immunosuppression minimization, withdrawal and tolerance induction may make living donation a desired first-line operation rather than a necessary albeit less-desirable option. This chapter summarizes the progress of living liver donation and its potential applications. PMID:23397534

  8. Living kidney donor experiences: implications for counselling.

    PubMed

    Walsh, A

    2004-01-01

    This study adds to previous, mostly quantitative, investigation into the experiences of living related kidney donors. Such investigation is important so that potential donors are supported effectively and donation programmes remain relevant and specific to need. Exploration takes place into donor decision-making processes and the most effective forms of professional support. A non-probability sampling technique highlighted eight living related kidney donors who were interviewed using a semi-structured interview format. Raw data was analysed through the qualitative technique of Interpretative Phenomenological Analysis. The decision to donate is made rapidly, decisively and rationally. Professional support provides reassurance to donors, particularly when experiencing acute psychological reaction. The need to provide support to the parents of living donors is specifically highlighted. A comprehensive range of Master Themes are generated through Interpretative Phenomenological Analysis and these reflect the complexity of the donation experience. This suggests donors are concerned with the management of psychological experience rather than with reviewing the appropriateness of an original decision to donate. Evidence indicates that concepts of attitude and self-efficacy belief can develop understanding of the psychological experience of being a living kidney donor. A counselling perspective, with Social Cognitive Theory at its core, is highlighted as a valid method for providing professional support to donors before and after surgery. PMID:15835410

  9. [Kidney transplant from living donors in children?].

    PubMed

    Ginevri, Fabrizio; Dello Strologo, Luca; Guzzo, Isabella; Belingheri, Mirco; Ghio, Luciana

    2011-01-01

    A living-donor kidney transplant offers a child at the terminal stages of renal disease better functional recovery and quality of life than an organ from a deceased donor. Before starting the procedure for a living-donor transplant, however, it is necessary to establish if it is really safe. There are diseases, such as focal segmental glomerulosclerosis, atypical HUS and membranoproliferative glomerulonephritis with dense deposits, for which living donation is not recommended given the high incidence of recurrence of the disease but also the frequent loss of the graft. Regarding the selection of the donor, an increased risk of acute rejection has been reported for donors older than 60-65 years and a worsening of the renal outcome if the donor's weight is equal to or less than the recipient's. Finally, it is necessary to take into consideration that complications may arise in the donor both in the perioperative period and in the long term. In conclusion, kidney transplant from a living donor is a natural choice within the pediatric setting. The parents, usually young and highly motivated to donate, are the ideal donors. However, although the risks associated with donation are minimal, they are not totally absent, and consequently it is mandatory to follow standardized procedures according to the guidelines issued by the Centro Nazionale Trapianti. PMID:21341241

  10. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides.

    PubMed

    Bergsma, Atze J; In 't Groen, Stijn Lm; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, Wwm Pim

    2016-01-01

    While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect. PMID:27623443

  11. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

    PubMed

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  12. DGTI Register of Rare Donors

    PubMed Central

    Hustinx, Hein

    2014-01-01

    Summary For patients with antibodies against the most common blood groups a rapid and efficient supply of compatible erythrocyte concentrates is self-evident. But typically we have to make the greatest effort providing blood for these patients, which have made antibodies against common blood groups. There are however patients with antibodies against rare blood group antigens that need special blood. The supply of such blood can be very difficult and mostly time-consuming. For this reason we set up a database of blood donors with rare blood groups. Since 2005 the BTS SRC Berne Ltd. has run this database on behalf of the Swiss BTS SRC. After a reorganization and extension of the database, conducted during 2011/2012, the data file was renamed ‘DGTI Register of Rare Donors’ and is now run under the patronage of the German Society for Transfusion Medicine and Immunohematology (DGTI). PMID:25538534

  13. [Liver transplants from living donors].

    PubMed

    Rogiers, X; Danninger, F; Malagó, M; Knoefel, W T; Gundlach, M; Bassas, A; Burdelski, M; Broelsch, C E

    1996-03-01

    In this article the authors discuss the advantages of Living Related Liver Transplantation (LRLT), criteria for the selection of donors and the standard operation technique. Among a total of 241 liver transplantation (LTx), 42 LRLT were performed at the University of Hamburg between October 1, 1991 and December 19, 1994. The body weight of recipients for LRLT ranged from 4,6 to 39 kg, with 64,2% having less than 10 kg. The volume of the donor left lateral liver lobe ranged from 100 cc to 350 cc. The average one year survival rate among electively operated patients-status 3-4 (UNOS 1995 classification) was 86.7%, two year survival rate 83.3%. The main advantages of LRLT are consired the following: 1. Absence of mortality on the waiting list, 2. Optimal timing of the transplantation (elective procedure, patient in a good condition), 3. Excellent organ (no primary non function), 4. A possible immunologic advantage, 5. Relief of the waiting list for cadaveric organs, 6. Psychological benefit for the family, 7. Cost effectiveness. Potential candidates for living donation with more than one cardiovascular risk factors were excluded. Social and psychological reasons leading to rejection of candidates were as follows: unstable family structure, expected professional or financial difficulties after living donation or withdrawal from consent. LRLT gives parents of a child with TLD a chance to avoid the risk of death on the waiting list or primary non function of the graft. LRLT has therefore established an important place in pediatric liver transplantation. PMID:8768973

  14. Polarization of human donor corneas.

    PubMed

    Parekh, Mohit; Ruzza, Alessandro; Ferrari, Stefano; Salvalaio, Gianni; Elbadawy, Hossein; Ponzin, Diego; Lipari, Eugenio

    2016-06-01

    To investigate the de-orientation effect of DSAEK grafts by observing the cross patterns and polarization power of human donor corneas using a polarizing device (Lumaxis(®)). Forty human donor corneas were placed in small petri-plates with epithelial side facing up. Polarizing power (arbitrary unit) and crosses were monitored and recorded by the software. The tissue was marked at 'Superior' position to ensure that the base and the polarizer are in alignment with each other after the cut. The anterior lamellar cut was performed using microkeratome. The lenticule was placed back in the same position as marked to mimic the alignment. The tissue was further rotated by 45° ensuring that the base of the cornea and the polarizer were in alignment. The polarization power and 'crosses' were identified at each step. The average of forty corneas from pre-cut to post-45° angular change showed statistically significant difference (p < 0.05) in terms of polarizing power. The cross-shaped pattern deformed and lost the sharpness towards 45° angle. However, multiple variances in terms of 'cross-patterns' were observed throughout the study. Lumaxis(®) was able to determine the worst quality tissue in terms of polarization (no black zone and crosses). Despite the quality of cross pattern which can be used as an additional objective parameter to evaluate the optical properties of the corneal tissue, this preliminary study needs to be further justified in terms of clinical relevance whether polarization changes with oriented or de-oriented grafts have any effects and consequences on the visual acuity. PMID:26920874

  15. Neural Differentiation Modulates the Vertebrate Brain Specific Splicing Program

    PubMed Central

    Madgwick, Alicia; Fort, Philippe; Hanson, Peter S.; Thibault, Philippe; Gaudreau, Marie-Claude; Lutfalla, Georges; Möröy, Tarik; Abou Elela, Sherif; Chaudhry, Bill; Elliott, David J.; Morris, Christopher M.; Venables, Julian P.

    2015-01-01

    Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation. PMID:25993117

  16. Multishot diffusion-weighted SPLICE PROPELLER MRI of the abdomen.

    PubMed

    Deng, Jie; Omary, Reed A; Larson, Andrew C

    2008-05-01

    Multishot FSE (fast spin echo)-based diffusion-weighted (DW)-PROPELLER (periodically rotated overlapping parallel lines with enhanced reconstruction) MRI offers the potential to reduce susceptibility artifacts associated with single-shot DW-EPI (echo-planar imaging) approaches. However, DW-PROPELLER in the abdomen is challenging due to the large field-of-view and respiratory motion during DW preparation. Incoherent signal phase due to motion will violate the Carr-Purcell-Meiboom-Gill (CPMG) conditions, leading to destructive interference between spin echo and stimulated echo signals and consequent signal cancellation. The SPLICE (split-echo acquisition of FSE signals) technique can mitigate non-CPMG artifacts in FSE-based sequences. For SPLICE, spin echo and stimulated echo are separated by using imbalanced readout gradients and extended acquisition window. Two signal families each with coherent phase properties are acquired at different intervals within the readout window. Separate reconstruction of these two signal families can avoid destructive phase interference. Phantom studies were performed to validate signal phase properties with different initial magnetization phases. This study evaluated the feasibility of combining SPLICE and PROPELLER for DW imaging of the abdomen. It is demonstrated that DW-SPLICE-PROPELLER can effectively mitigate non-CPMG artifacts and improve DW image quality and apparent diffusion coefficient (ADC) map homogeneity. PMID:18429036

  17. RNA catalyzes nuclear pre-mRNA splicing

    PubMed Central

    Fica, Sebastian M.; Tuttle, Nicole; Novak, Thaddeus; Li, Nan-Sheng; Lu, Jun; Koodathingal, Prakash; Dai, Qing; Staley, Jonathan P.; Piccirilli, Joseph A.

    2014-01-01

    SUMMARY In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a multi-megadalton machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, following the discovery of self-splicing group II intron RNAs, the snRNAs were hypothesized to catalyze splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported to date. By using metal rescue strategies, here we show that the U6 snRNA catalyzes both splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Strikingly, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms, and likely common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome. PMID:24196718

  18. ERRβ splice variants differentially regulate cell cycle progression

    PubMed Central

    Heckler, Mary Mazzotta; Riggins, Rebecca B

    2015-01-01

    Orphan receptors comprise nearly half of all members of the nuclear receptor superfamily. Despite having broad structural similarities to the classical estrogen receptors, estrogen-related receptors (ERRs) have their own unique DNA response elements and functions. In this study, we focus on 2 ERRβ splice variants, short form ERRβ (ERRβsf) and ERRβ2, and identify their differing roles in cell cycle regulation. Using DY131 (a synthetic agonist of ERRβ), splice-variant selective shRNA, and exogenous ERRβsf and ERRβ2 cDNAs, we demonstrate the role of ERRβsf in mediating the G1 checkpoint through p21. We also show ERRβsf is required for DY131-induced cellular senescence. A key novel finding of this study is that ERRβ2 can mediate a G2/M arrest in response to DY131. In the absence of ERRβ2, the DY131-induced G2/M arrest is reversed, and this is accompanied by p21 induction and a G1 arrest. This study illustrates novel functions for ERRβ splice variants and provides evidence for splice variant interaction. PMID:25496115

  19. Conserved mechanism of tRNA splicing in eukaryotes.

    PubMed Central

    Zillmann, M; Gorovsky, M A; Phizicky, E M

    1991-01-01

    The ligation steps of tRNA splicing in yeast and vertebrate cells have been thought to proceed by fundamentally different mechanisms. Ligation in yeast cells occurs by incorporation of an exogenous phosphate from ATP into the splice junction, with concomitant formation of a 2' phosphate at the 5' junction nucleotide. This phosphate is removed in a subsequent step which, in vitro, is catalyzed by an NAD-dependent dephosphorylating activity. In contrast, tRNA ligation in vertebrates has been reported to occur without incorporation of exogenous phosphate or formation of a 2' phosphate. We demonstrate in this study the existence of a yeast tRNA ligase-like activity in HeLa cells. Furthermore, in extracts from these cells, the entire yeastlike tRNA splicing machinery is intact, including that for cleavage, ligation, and removal of the 2' phosphate in an NAD-dependent fashion to give mature tRNA. These results argue that the mechanism of tRNA splicing is conserved among eukaryotes. Images PMID:1922054

  20. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1)....

  1. Leveraging transcript quantification for fast computation of alternative splicing profiles.

    PubMed

    Alamancos, Gael P; Pagès, Amadís; Trincado, Juan L; Bellora, Nicolás; Eyras, Eduardo

    2015-09-01

    Alternative splicing plays an essential role in many cellular processes and bears major relevance in the understanding of multiple diseases, including cancer. High-throughput RNA sequencing allows genome-wide analyses of splicing across multiple conditions. However, the increasing number of available data sets represents a major challenge in terms of computation time and storage requirements. We describe SUPPA, a computational tool to calculate relative inclusion values of alternative splicing events, exploiting fast transcript quantification. SUPPA accuracy is comparable and sometimes superior to standard methods using simulated as well as real RNA-sequencing data compared with experimentally validated events. We assess the variability in terms of the choice of annotation and provide evidence that using complete transcripts rather than more transcripts per gene provides better estimates. Moreover, SUPPA coupled with de novo transcript reconstruction methods does not achieve accuracies as high as using quantification of known transcripts, but remains comparable to existing methods. Finally, we show that SUPPA is more than 1000 times faster than standard methods. Coupled with fast transcript quantification, SUPPA provides inclusion values at a much higher speed than existing methods without compromising accuracy, thereby facilitating the systematic splicing analysis of large data sets with limited computational resources. The software is implemented in Python 2.7 and is available under the MIT license at https://bitbucket.org/regulatorygenomicsupf/suppa. PMID:26179515

  2. Coordinated Dynamics of RNA Splicing Speckles in the Nucleus.

    PubMed

    Zhang, Qiao; Kota, Krishna P; Alam, Samer G; Nickerson, Jeffrey A; Dickinson, Richard B; Lele, Tanmay P

    2016-06-01

    Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces. PMID:26496460

  3. Deep learning of the tissue-regulated splicing code

    PubMed Central

    Leung, Michael K. K.; Xiong, Hui Yuan; Lee, Leo J.; Frey, Brendan J.

    2014-01-01

    Motivation: Alternative splicing (AS) is a regulated process that directs the generation of different transcripts from single genes. A computational model that can accurately predict splicing patterns based on genomic features and cellular context is highly desirable, both in understanding this widespread phenomenon, and in exploring the effects of genetic variations on AS. Methods: Using a deep neural network, we developed a model inferred from mouse RNA-Seq data that can predict splicing patterns in individual tissues and differences in splicing patterns across tissues. Our architecture uses hidden variables that jointly represent features in genomic sequences and tissue types when making predictions. A graphics processing unit was used to greatly reduce the training time of our models with millions of parameters. Results: We show that the deep architecture surpasses the performance of the previous Bayesian method for predicting AS patterns. With the proper optimization procedure and selection of hyperparameters, we demonstrate that deep architectures can be beneficial, even with a moderately sparse dataset. An analysis of what the model has learned in terms of the genomic features is presented. Contact: frey@psi.toronto.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24931975

  4. Stiff, Strong Splice For A Composite Sandwich Structure

    NASA Technical Reports Server (NTRS)

    Schmaling, D.

    1991-01-01

    New type of splice for composite sandwich structure reduces peak shear stress in structure. Layers of alternating fiber orientation interposed between thin ears in adhesive joint. Developed for structural joint in spar of helicopter rotor blade, increases precision of control over thickness of adhesive at joint. Joint easy to make, requires no additional pieces, and adds little weight.

  5. The reciprocal regulation between splicing and 3'-end processing.

    PubMed

    Kaida, Daisuke

    2016-07-01

    Most eukaryotic precursor mRNAs are subjected to RNA processing events, including 5'-end capping, splicing and 3'-end processing. These processing events were historically studied independently; however, since the early 1990s tremendous efforts by many research groups have revealed that these processing factors interact with each other to control each other's functions. U1 snRNP and its components negatively regulate polyadenylation of precursor mRNAs. Importantly, this function is necessary for protecting the integrity of the transcriptome and for regulating gene length and the direction of transcription. In addition, physical and functional interactions occur between splicing factors and 3'-end processing factors across the last exon. These interactions activate or inhibit splicing and 3'-end processing depending on the context. Therefore, splicing and 3'-end processing are reciprocally regulated in many ways through the complex protein-protein interaction network. Although interesting questions remain, future studies will illuminate the molecular mechanisms underlying the reciprocal regulation. WIREs RNA 2016, 7:499-511. doi: 10.1002/wrna.1348 For further resources related to this article, please visit the WIREs website. PMID:27019070

  6. Neural differentiation modulates the vertebrate brain specific splicing program.

    PubMed

    Madgwick, Alicia; Fort, Philippe; Hanson, Peter S; Thibault, Philippe; Gaudreau, Marie-Claude; Lutfalla, Georges; Möröy, Tarik; Abou Elela, Sherif; Chaudhry, Bill; Elliott, David J; Morris, Christopher M; Venables, Julian P

    2015-01-01

    Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation. PMID:25993117

  7. Electromechanical behaviour of REBCO tape lap splices under transverse compressive loading

    NASA Astrophysics Data System (ADS)

    Grether, A.; Scheuerlein, C.; Ballarino, A.; Bottura, L.

    2016-07-01

    We have studied the influence of transverse compressive stress on the resistance and critical current (I c ) of soldered REBCO tape lap splices. Internal contact resistances dominate the overall REBCO lap splice resistances. Application of transverse compressive stress up to 250 MPa during the resistance measurements does not alter the resistance and I c of the soldered REBCO splices that were studied. The resistance of unsoldered REBCO tape lap splices depends strongly on the contact pressure. At a transverse compressive stress of 100 MPa, to which Roebel cables are typically exposed in high field magnets, the crossover splice contact resistance is comparable to the internal tape resistances.

  8. AVISPA: a web tool for the prediction and analysis of alternative splicing.

    PubMed

    Barash, Yoseph; Vaquero-Garcia, Jorge; González-Vallinas, Juan; Xiong, Hui Yuan; Gao, Weijun; Lee, Leo J; Frey, Brendan J

    2013-01-01

    Transcriptome complexity and its relation to numerous diseases underpins the need to predict in silico splice variants and the regulatory elements that affect them. Building upon our recently described splicing code, we developed AVISPA, a Galaxy-based web tool for splicing prediction and analysis. Given an exon and its proximal sequence, the tool predicts whether the exon is alternatively spliced, displays tissue-dependent splicing patterns, and whether it has associated regulatory elements. We assess AVISPA's accuracy on an independent dataset of tissue-dependent exons, and illustrate how the tool can be applied to analyze a gene of interest. AVISPA is available at http://avispa.biociphers.org. PMID:24156756

  9. Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism

    USGS Publications Warehouse

    Ramirez-Gomez, F.; Greene, W.; Rego, K.; Hansen, J.D.; Costa, G.; Kataria, P.; Bromage, E.S.

    2012-01-01

    The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cμ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD+ plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

  10. Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

    PubMed Central

    Leprince, D; Duterque-Coquillaud, M; Li, R P; Henry, C; Flourens, A; Debuire, B; Stehelin, D

    1988-01-01

    Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence. Images PMID:2841475

  11. Alternative Splice Transcripts for MHC Class I-like MICA Encode Novel NKG2D Ligands with Agonist or Antagonist Functions.

    PubMed

    Gavlovsky, Pierre-Jean; Tonnerre, Pierre; Gérard, Nathalie; Nedellec, Steven; Daman, Andrew W; McFarland, Benjamin J; Charreau, Béatrice

    2016-08-01

    MHC class I chain-related proteins A and B (MICA and MICB) and UL16-binding proteins are ligands of the activating NKG2D receptor involved in cancer and immune surveillance of infection. Structurally, MICA/B proteins contain an α3 domain, whereas UL16-binding proteins do not. We identified novel alternative splice transcripts for MICA encoding five novel MICA isoforms: MICA-A, -B1, -B2, -C, and -D. Alternative splicing associates with MICA*015 and *017 and results from a point deletion (G) in the 5' splice donor site of MICA intron 4 leading to exon 3 and exon 4 skipping and/or deletions. These changes delete the α3 domain in all isoforms, and the α2 domain in the majority of isoforms (A, B1, C, and D). Endothelial and hematopoietic cells contained endogenous alternative splice transcripts and isoforms. MICA-B1, -B2, and -D bound NKG2D by surface plasmon resonance and were expressed at the cell surface. Functionally, MICA-B2 contains two extracellular domains (α1 and α2) and is a novel potent agonist ligand for NKG2D. We found that MICA-D is a new truncated form of MICA with weak affinity for NKG2D despite lacking α2 and α3 domains. MICA-D may functionally impair NKG2D activation by competing with full-length MICA or MICA-B2 for NKG2D engagement. Our study established NKG2D binding for recombinant MICA-B1 but found no function for this isoform. New truncated MICA isoforms exhibit a range of functions that may drive unexpected immune mechanisms and provide new tools for immunotherapy. PMID:27342847

  12. The DEAH-box ATPases Prp16 and Prp43 cooperate to proofread 5′ splice site cleavage during pre-mRNA splicing

    PubMed Central

    Koodathingal, Prakash; Novak, Thaddeus; Piccirilli, Joseph A.; Staley, Jonathan P.

    2010-01-01

    SUMMARY To investigate the mechanism of fidelity in pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5′ splice site cleavage. We inactivated spliceosomes by disrupting a critical metal-ligand interaction at the catalytic center and discovered that when the DEAH-box ATPase Prp16 was disabled these spliceosomes successfully catalyzed 5′ splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5′ splice site cleavage, we found that Prp16 can associate with spliceosomes before 5′ splice site cleavage, consistent with a role for Prp16 in proofreading 5′ splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH-box ATPase Prp43, a spliceosome disassembly factor. These data provide evidence that splicing fidelity mechanisms discriminate against slow substrates and that the mechanisms for establishing the fidelity of 5′ splice site cleavage and exon ligation share a common ATP-dependent framework. PMID:20705241

  13. The Experience of Living Kidney Donors

    ERIC Educational Resources Information Center

    Brown, Judith Belle; Karley, Mary Lou; Boudville, Neil; Bullas, Ruth; Garg, Amit X.; Muirhead, Norman

    2008-01-01

    This article describes the experiences, feelings, and ideas of living kidney donors. Using a phenomenological, qualitative research approach, the authors interviewed 12 purposefully selected living kidney donors (eight men and four women), who were between four and 29 years since donation. Interviews were audiotaped, and transcribed verbatim, and…

  14. The value of living donor liver transplantation.

    PubMed

    Yang, Xiaoli; Gong, Junhua; Gong, JianPing

    2012-12-31

    Living donor liver transplantation (LDLT) is a very successful procedure that develops liver resources in case of worldwide shortages. As the technology has developed so much in the past 2 decades, LDLT has the same good prognosis as DDLT. However, LDLT still has lots of ethical & technical problems. It causes great psychiatric, physical and psychosocial harm to donors. Also, it has some negative effects on society by providing a platform for organ trade. Therefore, there is much controversy about the social value of LDLT. After review of recent papers, we find much progress can be made in inspiring the public to become organ donors and creating donation model new to improve the consent rate for solid organ donation from deceased donors. That is the key strategy for increasing the liver supply. With this serious shortage of organs, liver donor transplantation still has its advantages, but we should not place all our hopes on LDLT to increase the liver supply. We all need to try our best to increase donor awareness and promote organ donor registration--when cadaver organs could meet the needs for liver transplantation, living donor liver transplants would not be necessary. PMID:23274332

  15. Payment for donor kidneys: pros and cons.

    PubMed

    Friedman, E A; Friedman, A L

    2006-03-01

    Continuous growth of the end stage renal disease population treated by dialysis, outpaces deceased donor kidneys available, lengthens the waiting time for a deceased donor transplant. As estimated by the United States Department of Health & Human Services: '17 people die each day waiting for transplants that can't take place because of the shortage of donated organs.' Strategies to expand the donor pool--public relations campaigns and Drivers' license designation--have been mainly unsuccessful. Although illegal in most nations, and viewed as unethical by professional medical organizations, the voluntary sale of purchased donor kidneys now accounts for thousands of black market transplants. The case for legalizing kidney purchase hinges on the key premise that individuals are entitled to control of their body parts even to the point of inducing risk of life. One approach to expanding the pool of kidney donors is to legalize payment of a fair market price of about 40,000 dollars to donors. Establishing a federal agency to manage marketing and purchase of donor kidneys in collaboration with the United Network for Organ Sharing might be financially self-sustaining as reduction in costs of dialysis balances the expense of payment to donors. PMID:16482095

  16. 42 CFR 35.64 - Donors.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Donors. 35.64 Section 35.64 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICAL CARE AND EXAMINATIONS HOSPITAL AND STATION MANAGEMENT Contributions for the Benefit of Patients § 35.64 Donors. Authorized contributions...

  17. 42 CFR 35.64 - Donors.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Donors. 35.64 Section 35.64 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICAL CARE AND EXAMINATIONS HOSPITAL AND STATION MANAGEMENT Contributions for the Benefit of Patients § 35.64 Donors. Authorized contributions...

  18. 42 CFR 35.64 - Donors.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Donors. 35.64 Section 35.64 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICAL CARE AND EXAMINATIONS HOSPITAL AND STATION MANAGEMENT Contributions for the Benefit of Patients § 35.64 Donors. Authorized contributions...

  19. Recipients' views on payment of sperm donors.

    PubMed

    Ravelingien, An; Provoost, Veerle; Wyverkens, Elia; Buysse, Ann; De Sutter, Petra; Pennings, Guido

    2015-08-01

    The aim of this qualitative study was to explore how recipients viewed payment of sperm donors. The study was conducted in Belgium, where, as in many countries, sperm donors receive recompense for their time and expenses. Face-to-face semi-structured interviews were conducted with 34 heterosexual and lesbian couples who, at the time of data collection, had at least one donor-conceived child aged 7-10 years or who were undergoing donor conception treatment. Although participants commonly described the issue of financial compensation as something that did not really concern them, all supported the idea that some level of payment was acceptable or even necessary. The participants also identified several ways in which donor payment offered advantages to their own position as (future) parents. Although the idea is commonly rehearsed that sperm donation is a gift and that monetary transaction for conception is demeaning, the participants of this study did not generally share this view. To them, a small financial return served as a symbolic acknowledgement of the donor's contribution and helped secure the type of relationship they expected from their donor. There was clearly concern, however, over high payments and the risk of attracting the wrong kind of donor. PMID:26099446

  20. Negotiating boundaries: Accessing donor gametes in India

    PubMed Central

    Widge, A.; Cleland, J.

    2011-01-01

    Background: This paper documents how couples and providers access donor materials for conception in the Indian context and perceptions about using them. The objective is to facilitate understanding of critical issues and relevant concerns. Methods: A postal survey was conducted with a sample of 6000 gynaecologists and in-depth interviews were conducted with 39 gynaecologists in four cities. Results: Donor gametes are relatively more acceptable than a few years ago, especially if confidentiality can be maintained, though lack of availability of donor materials is sometimes an impediment to infertility treatment. Donor sperms are usually accessed from in-house or commercial sperm banks, pathology laboratories, IVF centres, professional donors, relatives or friends. There is scepticism about screening procedures of sperm banks. Donor eggs are usually accessed from voluntary donors, friends, relatives, egg sharing programmes, donation from other patients, advertising and commercial donors. There are several concerns regarding informed consent for using donated gametes, using relatives and friends gametes, the unregulated use of gametes and embryos, record keeping and documentation, unethical and corrupt practices and commercialisation. Conclusion: These issues need to be addressed by patients, providers and regulatory authorities by providing information, counselling, ensuring informed consent, addressing exploitation and commercialisation, ensuring monitoring, proper documentation and transparency. PMID:24753849