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1

Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site.  

PubMed Central

In Caenorhabditis elegans, pre-mRNAs that are trans-spliced are distinguished by the presence of an 'outron', intron-like RNA at the 5' end followed by a splice acceptor. We report that trans-splicing of the rol-6 gene can be completely suppressed simply by introducing a donor site into its 173 nt outron, at a site 50 nt upstream of the trans-splice site, thereby converting rol-6 into a conventional gene with a spliced intron near its 5' end. When the consensus donor site was inserted at sites further upstream it was less effective in replacing transplicing with cis-splicing. Surprisingly, the length of the intron was not the important variable, since lengthening of the 50 nt intron to 250 nt did not restore trans-splicing. Apparently the context into which the splice site was introduced determined the efficiency of its use. These results support the conclusion that the sole signal for trans-splicing is the presence of an outron. Clearly, cis- and trans-splice acceptor sites are interchangeable, allowing the possibility of competition between the two types of splicing. Images

Conrad, R; Liou, R F; Blumenthal, T

1993-01-01

2

Low U1 snRNP dependence at the NF1 exon 29 donor splice site.  

PubMed

Many disease-causing splicing mutations described in the literature produce changes in splice sites (SS) or in exon-regulatory sequences. The delineation of these splice aberrations can provide important insights into novel regulation mechanisms. In this study, we evaluated the effect of patient variations in neurofibromatosis type 1 (NF1) exon 29 and its 5'SS surrounding area on its splicing process. Only two of all nonsense, missense, synonymous and intronic variations analyzed in this study clearly altered exon 29 inclusion/exclusion levels. In particular, the intronic mutation +5g>a had the strongest effect, resulting in total exon exclusion. This finding prompted us to evaluate the exon 29 5'SS in relation to its ability to bind U1 snRNP. This was performed by direct analysis of the ability of U1 to bind to wild-type and mutant donor sites, by engineering an in vitro splicing system to directly evaluate the functional importance of U1 snRNA base pairing with the exon 29 donor site, and by coexpression of mutant U1 snRNP molecules to try to rescue exon 29 inclusion in vivo. The results revealed a low dependency on the presence of U1 snRNP, and suggest that exon 29 donor site definition may depend on alternative mechanisms of 5'SS recognition. PMID:19292874

Raponi, Michela; Buratti, Emanuele; Dassie, Elisa; Upadhyaya, Meena; Baralle, Diana

2009-04-01

3

Antisense suppression of donor splice site mutations in the dystrophin gene transcript  

PubMed Central

We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis.

Fletcher, Sue; Meloni, Penny L; Johnsen, Russell D; Wong, Brenda L; Muntoni, Francesco; Wilton, Stephen D

2013-01-01

4

Sequence contexts that determine the pathogenicity of base substitutions at position +3 of donor splice-sites  

Microsoft Academic Search

Variations at position 13 of 50 splice-sites (50ss) are reported to induce aberrant splicing in some cases but not in others suggesting that the overall nucleotidic environment can dictate the extent to which 50ss are correctly selected. Functional studies of three variations identified in donor splice-sites of USH2A and PCDH15 genes sustain this assumption. To gain insights into this question,

Nicolas Molinari; Julie Vaudaine; Mireille Claustres; Sylvie Tuffery-Giraud

2009-01-01

5

Prenatal diagnosis and a donor splice site mutation in fibrillin in a family with Marfan syndrome  

PubMed Central

The Marfan syndrome, an autosomal dominant connective tissue disorder, is manifested by abnormalities in the cardiovascular, skeletal, and ocular systems. Recently, fibrillin, an elastin-associated microfibrillar glycoprotein, has been linked to the Marfan syndrome, and fibrillin mutations in affected individuals have been documented. In this study, genetic linkage analysis with fibrillin specific markers was used to establish the prenatal diagnosis in an 11-wk-gestation fetus in a four-generation Marfan kindred. At birth, skeletal changes suggestive of the Marfan syndrome were observed. Reverse transcription–PCR amplification of the fibrillin gene mRNA detected a deletion of 123 bp in one allele in affected relatives. This deletion corresponds to an exon encoding an epidermal growth factor–like motif. Examination of genomic DNA showed a G?C transversion at the +1 consensus donor splice site. ImagesFigure 3Figure 4Figure 5Figure 6Figure 7

Godfrey, Maurice; Vandemark, Natalie; Wang, Mei; Velinov, Milen; Wargowski, David; Tsipouras, Petros; Han, Jenny; Becker, Joanne; Robertson, Wendy; Droste, Sabine; Rao, Velidi H.

1993-01-01

6

Prenatal diagnosis and a donor splice site mutation in fibrillin in a family with Marfan syndrome  

SciTech Connect

The Marfan syndrome, an autosomal dominant connective tissue disorder, is manifested by abnormalities in the cardiovascular, skeletal, and ocular systems. Recently, fibrillin, an elastic-associated microfibrillar glycoprotein, has been linked to the Marfan syndrome, and fibrillin mutations in affected individuals have been documented. In this study, genetic linkage analysis with fibrillin-specific markers was used to establish the prenatal diagnosis in an 11-wk-gestation fetus in a four-generation Marfan kindred. At birth, skeletal changes suggestive of the Marfan syndrome were observed. Reverse transcription-PCR amplification of the fibrillin gene mRNA detected a deletion of 123 bp in one allele in affected relatives. This deletion corresponds to an exon encoding an epidermal growth factor-like motif. Examination of genomic DNA showed a G[yields]C transversion at the +1 consensus donor splice site. 45 refs., 7 figs.

Godfrey, M.; Vandemark, N.; Wang, M.; Han, J.; Rao, V.H. (Univ. of Nebraska Medical Center, Omaha (United States)); Velinov, M.; Tsipouras, P. (Univ. of Connecticut Health Sciences Center, Farmington (United States)); Wargowski, D.; Becker, J.; Robertson, W.; Droste, S. (Univ. of Wisconsin, Madison (United States))

1993-08-01

7

Novel Donor Splice Site Mutation of ABCG5 Gene in Sitosterolemia  

Microsoft Academic Search

In a patient with sitosterolemia, we found two different mutations of the ATP-binding cassette, subfamily G, member 5 (ABCG5) gene. The first is a missense mutation that changes the amino acid residue at position 419 from arginine to histidine, i.e., R419H. The second is a novel splicing mutation affecting the invariant guanine at the first base of the donor splice

Ching-Wan Lam; Anna Wai-Fun Cheng; Sui-Fan Tong; Yan-Wo Chan

2002-01-01

8

Two alpha subunit donor splice site mutations cause human trifunctional protein deficiency.  

PubMed Central

Human trifunctional protein catalyzes three steps in mitochondrial beta-oxidation of fatty acids, including the long chain 3-hydroxyacyl-CoA dehydrogenase step. Deficiency of this heterocomplex, which contains 4 alpha and 4 beta subunits, causes sudden unexplained infant death, a Reye-like syndrome, cardiomyopathy, or skeletal myopathy. We determined the molecular basis of this deficiency in a patient with neonatal presentation and later sudden death using reverse transcription and PCR amplification of his alpha subunit mRNA. We demonstrated a universal deletion of exon 3 (71 bp) in his mRNA. This deletion causes a frameshift and very early premature termination. Amplification of genomic DNA demonstrated that the patient was a compound heterozygote with two different mutations in the 5' donor splice site following exon 3: a paternally inherited G to A transversion at the invariant position +1 and a maternally inherited A to G mutation at position +3. Both allelic mutations apparently cause exon 3 skipping, resulting in undetectable levels of alpha subunit protein, and complete loss of trifunctional protein. This is the initial molecular characterization of trifunctional protein deficiency. Images

Brackett, J C; Sims, H F; Rinaldo, P; Shapiro, S; Powell, C K; Bennett, M J; Strauss, A W

1995-01-01

9

The intron 7 donor splice site transition: a second Tay-Sachs disease mutation in French Canada  

Microsoft Academic Search

Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a G?A transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts

Peter Hechtman; Bernard Boulay; Marc De Braekeleer; Eve Andermann; Serge Melançon; Jean Larochelle; Claude Prevost; Feige Kaplan

1992-01-01

10

The HIV-1 5' LTR poly(A) site is inactivated by U1 snRNP interaction with the downstream major splice donor site.  

PubMed Central

The inactivity of the 5' long terminal repeat (LTR) poly(A) site immediately downstream of the cap site maximizes the production of HIV-1 transcripts. In this paper, we demonstrate that this inactivity is mediated by the interaction of the U1 snRNP with the major splice donor site (MSD). The inhibition of the HIV-1 poly(A) site by U1 snRNP relies on a series of delicately balanced RNA processing signals. These include the poly(A) site, the major splice donor site and the splice acceptor sites. The inherent efficiency of the HIV-1 poly(A) site allows maximal activity where there is no donor site (in the 3' LTR) but full inhibition by the downstream MSD (in the 5' LTR). The MSD must interact efficiently with U1 snRNP to completely inhibit the 5' LTR poly(A) site, whereas the splice acceptor sites are inefficient, allowing full-length genomic RNA production.

Ashe, M P; Pearson, L H; Proudfoot, N J

1997-01-01

11

Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.  

PubMed Central

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested. Images

Mayeda, A; Ohshima, Y

1988-01-01

12

Further investigation of the HEXA gene intron 9 donor splice site mutation frequently found in non-Jewish Tay-Sachs disease patients from the British Isles  

Microsoft Academic Search

In a previous study we found that a Tay-Sachs disease (TSD) causing mutation in the intron 9 donor splice site of the HEXA gene occurs at high frequency in non-Jewish patients and carriers from the British Isles. It was found more frequently in subjects of Irish, Scottish, and Welsh origin compared with English origin (63% and 31% respectively). We have

E C Landels; P M Green; I H Ellis; A H Fensom; M M Kaback; J Lim-Steele; K Zeiger; N Levy; M Bobrow

1993-01-01

13

RNA Structure Modulates Splicing Efficiency at the Human Immunodeficiency Virus Type 1 Major Splice Donor  

Microsoft Academic Search

The untranslated leader of the human immunodeficiency virus type 1 (HIV-1) RNA genome encodes essential sequence and structural motifs that control various replication steps. The 5 splice site or splice donor (SD) is embedded in a semistable hairpin, but the function of this structure is unknown. We stabilized this SD hairpin by creating an additional base pair and demonstrated a

Truus E. M. Abbink; Ben Berkhout

2008-01-01

14

Insertion of a T next to the donor splice site of intron 1 causes aberrantly spliced mRNA in a case of infantile GM1-gangliosidosis.  

PubMed

The lysosomal storage disorders GM1-gangliosidosis and Morquio B syndrome are caused by a complete or partial deficiency of acid beta-galactosidase. Here, we have characterized the mutation segregating in a family with two siblings affected by the severe infantile form of GM1-gangliosidosis. In total mRNA preparations derived from the patients' fibroblasts at least two aberrantly spliced beta-galactosidase transcripts (1 and 2) have been identified. Both transcripts contain a 20 nucleotide (nt) insertion derived from the 5' end of intron 1 of the beta-galactosidase gene. Furthermore, in transcript 2 sequences encoded by exon II are deleted during the splicing process. Comparison of the 20-nt insertion with wild-type intronic sequences indicated that in the genomic DNA of the patients an extra T nucleotide is present immediately downstream of the conserved GT splice donor dinucleotide of intron 1. Both patients are homozygous for the T nucleotide insertion. We propose that this single base insertion is the mutation responsible for aberrant splicing of beta-galactosidase pre-mRNA, giving rise to transcripts that cannot encode a normal protein. PMID:8199591

Morrone, A; Morreau, H; Zhou, X Y; Zammarchi, E; Kleijer, W J; Galjaard, H; d'Azzo, A

1994-01-01

15

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

PubMed Central

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.

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

16

Alternative splicing regulation at tandem 3? splice sites  

PubMed Central

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, ?25% are related to alternative choices of 3? and 5? splice sites. Surprisingly, half of all these events involve 3? splice sites that are exactly 3 nt apart. These tandem 3? splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of ?30 bp at the intronic regions flanking the 3? splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.

Akerman, Martin; Mandel-Gutfreund, Yael

2006-01-01

17

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

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.

Solera, J. (Unidades de Genetica Molecular, Madrid (Spain)); Magallon, M.; Martin-Villar, J. (Hemofilia Hospital, Madrid (Spain)); Coloma, A. (Departamento deBioquimica de la Facultad de Medicina de la Universidad Autonoma, Madrid (Spain))

1992-02-01

18

Identification of alternative 5?/3? splice sites based on the mechanism of splice site competition  

PubMed Central

Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5?/3? splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified ?70% of the splice sites into alternative and constitutive, as well as ?80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology.

Xia, Huiyu; Bi, Jianning; Li, Yanda

2006-01-01

19

Alternative 5' splice site selection induced by heat shock.  

PubMed Central

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression. Images

Takechi, H; Hosokawa, N; Hirayoshi, K; Nagata, K

1994-01-01

20

Prediction of human mRNA donor and acceptor sites from the DNA sequence*1  

Microsoft Academic Search

Artificial neural networks have been applied to the prediction of splice site location in humanpre--mRNA. A joint prediction scheme where prediction of transition regions between intronsand exons regulates a cutoff level for splice site assignment was able to predict splice sitelocations with confidence levels far better than previously reported in the literature. Theproblem of predicting donor and acceptor sites in

Søren Brunak; Jacob Engelbrecht; Steen Knudsen

1991-01-01

21

Feature subset selection for splice site prediction  

Microsoft Academic Search

Motivation: The large amount of available annotated Arabidopsis thaliana sequences allows the induction of splice site prediction models with supervised learning algorithms (see Haussler (1998) for a review and refer- ences). These algorithms need information sources or features from which the models can be computed. For splice site prediction, the features we consider in this study are the presence or

Sven Degroeve; Bernard De Baets; Yves Van De Peer; Pierre Rouzé

2002-01-01

22

Comparison of splice sites in mammals and chicken  

PubMed Central

We have carried out an initial analysis of the dynamics of the recent evolution of the splice-sites sequences on a large collection of human, rodent (mouse and rat), and chicken introns. Our results indicate that the sequences of splice sites are largely homogeneous within tetrapoda. We have also found that orthologous splice signals between human and rodents and within rodents are more conserved than unrelated splice sites, but the additional conservation can be explained mostly by background intron conservation. In contrast, additional conservation over background is detectable in orthologous mammalian and chicken splice sites. Our results also indicate that the U2 and U12 intron classes seem to have evolved independently since the split of mammals and birds; we have not been able to find a convincing case of interconversion between these two classes in our collections of orthologous introns. Similarly, we have not found a single case of switching between AT-AC and GT-AG subtypes within U12 introns, suggesting that this event has been a rare occurrence in recent evolutionary times. Switching between GT-AG and the noncanonical GC-AG U2 subtypes, on the contrary, does not appear to be unusual; in particular, T to C mutations appear to be relatively well tolerated in GT-AG introns with very strong donor sites.

Abril, Josep F.; Castelo, Robert; Guigo, Roderic

2005-01-01

23

Unusual splice site mutations disrupt FANCA exon 8 definition.  

PubMed

The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position -3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition. PMID:24704046

Mattioli, Chiara; Pianigiani, Giulia; De Rocco, Daniela; Bianco, Anna Monica Rosaria; Cappelli, Enrico; Savoia, Anna; Pagani, Franco

2014-07-01

24

Fast splice site detection using information content and feature reduction  

PubMed Central

Background Accurate identification of splice sites in DNA sequences plays a key role in the prediction of gene structure in eukaryotes. Already many computational methods have been proposed for the detection of splice sites and some of them showed high prediction accuracy. However, most of these methods are limited in terms of their long computation time when applied to whole genome sequence data. Results In this paper we propose a hybrid algorithm which combines several effective and informative input features with the state of the art support vector machine (SVM). To obtain the input features we employ information content method based on Shannon's information theory, Shapiro's score scheme, and Markovian probabilities. We also use a feature elimination scheme to reduce the less informative features from the input data. Conclusion In this study we propose a new feature based splice site detection method that shows improved acceptor and donor splice site detection in DNA sequences when the performance is compared with various state of the art and well known methods.

Baten, AKMA; Halgamuge, SK; Chang, BCH

2008-01-01

25

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

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.

Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X. [Cancer Research Institute, Barcelona (Spain); Doerk, T.; Will, K. [Medizinische Hochschule Hannover (Germany); Fonknechten, N. [Institut Cochin de Genetique Moleculaire, Paris (France)

1995-03-01

26

The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.  

PubMed Central

Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCKB receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome 11p15.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 amino acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin- and CCK-mediated signal transduction. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5

Song, I; Brown, D R; Wiltshire, R N; Gantz, I; Trent, J M; Yamada, T

1993-01-01

27

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

PubMed Central

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

Tigges, M A; Raskas, H J

1984-01-01

28

The human XPC DNA repair gene: arrangement, splice site information content and influence of a single nucleotide polymorphism in a splice acceptor site on alternative splicing and function  

PubMed Central

XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82–882 bp) and 15 introns (0.08–5.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcription–polymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that skipped exons 4, 7 or 12. Exon 7 was skipped in ?0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in ?0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (–0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (?2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility.

Khan, Sikandar G.; Muniz-Medina, Vanessa; Shahlavi, Tala; Baker, Carl C.; Inui, Hiroki; Ueda, Takahiro; Emmert, Steffen; Schneider, Thomas D.; Kraemer, Kenneth H.

2002-01-01

29

iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition  

PubMed Central

In eukaryotic genes, exons are generally interrupted by introns. Accurately removing introns and joining exons together are essential processes in eukaryotic gene expression. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapid and effective detection of splice sites that play important roles in gene structure annotation and even in RNA splicing. Although a series of computational methods were proposed for splice site identification, most of them neglected the intrinsic local structural properties. In the present study, a predictor called “iSS-PseDNC” was developed for identifying splice sites. In the new predictor, the sequences were formulated by a novel feature-vector called “pseudo dinucleotide composition” (PseDNC) into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on two benchmark datasets that the overall success rates achieved by iSS-PseDNC in identifying splice donor site and splice acceptor site were 85.45% and 87.73%, respectively. It is anticipated that iSS-PseDNC may become a useful tool for identifying splice sites and that the six DNA local structural properties described in this paper may provide novel insights for in-depth investigations into the mechanism of RNA splicing.

Feng, Peng-Mian; Chou, Kuo-Chen

2014-01-01

30

iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition.  

PubMed

In eukaryotic genes, exons are generally interrupted by introns. Accurately removing introns and joining exons together are essential processes in eukaryotic gene expression. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapid and effective detection of splice sites that play important roles in gene structure annotation and even in RNA splicing. Although a series of computational methods were proposed for splice site identification, most of them neglected the intrinsic local structural properties. In the present study, a predictor called "iSS-PseDNC" was developed for identifying splice sites. In the new predictor, the sequences were formulated by a novel feature-vector called "pseudo dinucleotide composition" (PseDNC) into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on two benchmark datasets that the overall success rates achieved by iSS-PseDNC in identifying splice donor site and splice acceptor site were 85.45% and 87.73%, respectively. It is anticipated that iSS-PseDNC may become a useful tool for identifying splice sites and that the six DNA local structural properties described in this paper may provide novel insights for in-depth investigations into the mechanism of RNA splicing. PMID:24967386

Chen, Wei; Feng, Peng-Mian; Lin, Hao; Chou, Kuo-Chen

2014-01-01

31

Comparative analysis of sequence features involved in the recognition of tandem splice sites  

PubMed Central

Background The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels of alternative tandem splice site exons occurring in different human organ systems and cell types, and to study known sequence features involved in the recognition and distinction of neighboring splice sites, we performed large-scale, stringent alignments of cDNA sequences and ESTs to the human and mouse genomes, followed by experimental validation. Results We analyzed alternative 5'ss exons (A5Es) and alternative 3'ss exons (A3Es), derived from transcript sequences that were aligned to assembled genome sequences to infer patterns of AS occurring in several thousands of genes. Comparing the levels of overlapping (tandem) and non-overlapping (competitive) A5Es and A3Es, a clear preference of isoforms was seen for tandem acceptors and donors, with four nucleotides and three to six nucleotides long exon extensions, respectively. A subset of inferred A5E tandem exons was selected and experimentally validated. With the focus on A5Es, we investigated their transcript coverage, sequence conservation and base-paring to U1 snRNA, proximal and distal splice site classification, candidate motifs for cis-regulatory activity, and compared A5Es with A3Es, constitutive and pseudo-exons, in H. sapiens and M. musculus. The results reveal a small but authentic enriched set of tandem splice site preference, with specific distances between proximal and distal 5'ss (3'ss), which showed a marked dichotomy between the levels of in- and out-of-frame splicing for A5Es and A3Es, respectively, identified a number of candidate NMD targets, and allowed a rough estimation of a number of undetected tandem donors based on splice site information. Conclusion This comparative study distinguishes tandem 5'ss and 3'ss, with three to six nucleotides long extensions, as having unusually high proportions of AS, experimentally validates tandem donors in a panel of different human tissues, highlights the dichotomy in the types of AS occurring at tandem splice sites, and elucidates that human alternative exons spliced at overlapping 5'ss posses features of typical splice variants that could well be beneficial for the cell.

Bortfeldt, Ralf; Schindler, Stefanie; Szafranski, Karol; Schuster, Stefan; Holste, Dirk

2008-01-01

32

The Birth of an Alternatively Spliced Exon: 3' Splice-Site Selection in Alu Exons  

Microsoft Academic Search

Alu repetitive elements can be inserted into mature messenger RNAs via a splicing-mediated process termed exonization. To understand the molecular basis and the regulation of the process of turning intronic Alus into new exons, we compiled and analyzed a data set of human exonized Alus. We revealed a mechanism that governs 3' splice-site selection in these exons during alternative splicing.

Galit Lev-Maor; Rotem Sorek; Noam Shomron; Gil Ast

2003-01-01

33

Characterisation of three novel splice site mutations in introns 11, 18 and 30 of the NF1 gene  

Microsoft Academic Search

Identification and characterization of germline mutations within the NF-1 gene was carried out in 25 unrelated NF-1 patients, in whom we have detected three splice site mutations which cause exon skipping. Our detection strategy incorporated both RNA and DNA as templates for PCR, chemical mismatch cleavage and direct sequencing. The first mutation was detected in the splice donor sequence of

S. M. Purandare; W. G. Lanyon; R. Arngrimsson

1994-01-01

34

Biological Dressings for Skin Graft Donor Sites.  

National Technical Information Service (NTIS)

Three methods of donor site management were tested in 17 patients to determine if any resulted in faster wound healing. Gross inspection and biopsies revealed no differences between donor sites left uncovered or those treated with fine mesh gauze. However...

B. A. Pruitt D. W. Wilmore P. Silverstein R. E. Salisbury

1972-01-01

35

Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.  

PubMed Central

The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs. Images

Aroian, R V; Levy, A D; Koga, M; Ohshima, Y; Kramer, J M; Sternberg, P W

1993-01-01

36

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals  

Microsoft Academic Search

BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1\\/A2

Daniel Gendron; Sandra Carriero; Daniel Garneau; Jonathan Villemaire; Roscoe Klinck; Sherif Abou Elela; Masad J Damha; Benoit Chabot

2006-01-01

37

Splice site identification using probabilistic parameters and SVM classification  

PubMed Central

Background Recent advances and automation in DNA sequencing technology has created a vast amount of DNA sequence data. This increasing growth of sequence data demands better and efficient analysis methods. Identifying genes in this newly accumulated data is an important issue in bioinformatics, and it requires the prediction of the complete gene structure. Accurate identification of splice sites in DNA sequences plays one of the central roles of gene structural prediction in eukaryotes. Effective detection of splice sites requires the knowledge of characteristics, dependencies, and relationship of nucleotides in the splice site surrounding region. A higher-order Markov model is generally regarded as a useful technique for modeling higher-order dependencies. However, their implementation requires estimating a large number of parameters, which is computationally expensive. Results The proposed method for splice site detection consists of two stages: a first order Markov model (MM1) is used in the first stage and a support vector machine (SVM) with polynomial kernel is used in the second stage. The MM1 serves as a pre-processing step for the SVM and takes DNA sequences as its input. It models the compositional features and dependencies of nucleotides in terms of probabilistic parameters around splice site regions. The probabilistic parameters are then fed into the SVM, which combines them nonlinearly to predict splice sites. When the proposed MM1-SVM model is compared with other existing standard splice site detection methods, it shows a superior performance in all the cases. Conclusion We proposed an effective pre-processing scheme for the SVM and applied it for the identification of splice sites. This is a simple yet effective splice site detection method, which shows a better classification accuracy and computational speed than some other more complex methods.

Baten, AKMA; Chang, BCH; Halgamuge, SK; Li, Jason

2006-01-01

38

New Splice Site Acceptor Mutation in AIRE Gene in Autoimmune Polyendocrine Syndrome Type 1  

PubMed Central

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison’s disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.

Mora, Mireia; Hanzu, Felicia A.; Pradas-Juni, Marta; Aranda, Gloria B.; Halperin, Irene; Puig-Domingo, Manuel; Aguilo, Sira; Fernandez-Rebollo, Eduardo

2014-01-01

39

Factors influencing alternative splice site utilization in vivo.  

PubMed Central

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

Fu, X Y; Manley, J L

1987-01-01

40

Splicing-coupled 3? end formation requires a terminal splice acceptor site, but not intron excision  

PubMed Central

Splicing of human pre-mRNA is reciprocally coupled to 3? end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3? end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated ?-globin gene. This is in part to alleviate the suppression of 3? end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3? end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3? end formation, but that on-going intron removal is less critical.

Davidson, Lee; West, Steven

2013-01-01

41

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites  

NASA Astrophysics Data System (ADS)

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Zuo, Ping; Manley, James L.

1994-04-01

42

Novel splice site mutation in keratin 1 underlies mild epidermolytic palmoplantar keratoderma in three kindreds.  

PubMed

We report a novel mutation in the exon 6 splice donor site of keratin 1 (G4134A) that segregates with a palmoplantar keratoderma in three kindreds. The nucleotide substitution leads to the utilization of a novel in-frame splice site 54 bases downstream of the mutation with the subsequent insertion of 18 amino acids into the 2B rod domain. This mutation appears to have a milder effect than previously described mutations in the helix initiation and termination sequence on the function of the rod domain, with regard to filament assembly and stability. Affected individuals displayed only mild focal epidermolysis in the spinous layer of palmoplantar epidermis, in comparison with cases of bullous congenital ichthyosiform erythroderma also due to keratin 1 mutations, which show widespread and severe epidermolysis. This study describes a novel mutation in KRT1 that results in a phenotype distinct from classical bullous congenital ichthyosiform erythroderma. PMID:11286630

Hatsell, S J; Eady, R A; Wennerstrand, L; Dopping-Hepenstal, P; Leigh, I M; Munro, C; Kelsell, D P

2001-04-01

43

Single-site retroperitoneoscopic donor nephrectomy.  

PubMed

We have performed retroperitoneoscopic nephrectomy for living kidney donor surgery since 2000. Recently, we introduced single-site retroperitoneoscopic donor nephrectomy (RDN) as a less invasive donor surgery. The procedure was performed in 7 donors (5 women and 2 men) by a single surgeon. The mean age and body mass index of the donors were 62.6 years (range, 53-74 years) and 24.3 kg/m(2) (range, 22.3-29.0 kg/m(2)), respectively. Left-sided nephrectomy was performed in all the donors. The donors were positioned in the right lateral position, and a 7-cm-long incision was made in the left flank. The incision was extended to the retroperitoneal space using the muscle-splitting technique. The retroperitoneal space was then expanded using an inflation balloon. A GelPOINT Advanced Access Platform (Applied Medical, Rancho Santa Margarita, Calif, United States) was placed in the incision. The subsequent technique and equipment were the same as those used in conventional 3-port RDN. The renal artery and vein were dissected using a vascular stapler, and the kidney graft was directly extracted through the incision. The mean operative time was 197 ± 28 minutes, warm ischemic time was 4.1 ± 1.2 minutes, and blood loss was 75 ± 113 mL. No statistical differences were found between the present method and conventional 3-port RDN. Intraoperative and postoperative complications were not observed in any of the donors. Graft function after transplantation was good, and delayed graft function was not observed in any of the recipients. This technique can be easily introduced in the clinical setting by surgeons experienced in RDN. PMID:24655953

Maruyama, M; Akutsu, N; Ohtsuki, K; Aoyama, H; Matsumoto, I; Hasegawa, M; Saigo, K; Asano, T

2014-03-01

44

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.  

PubMed Central

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence. Images

Carothers, A M; Urlaub, G; Grunberger, D; Chasin, L A

1993-01-01

45

Extended base pair complementarity between U1 snRNA and the 50 splice site does not inhibit splicing in higher eukaryotes, but rather increases 50 splice site recognition  

Microsoft Academic Search

Spliceosome formation is initiated by the recognition of the 50 splice site through formation of an RNA duplex between the 50 splice site and U1 snRNA. We have previously shown that RNA duplex format- ion between U1 snRNA and the 50 splice site can pro- tect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to

Marcel Freund; Martin J. Hicks; Carolin Konermann; Marianne Otte; Klemens J. Hertel; Heiner Schaal

2005-01-01

46

An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.  

PubMed Central

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.

Chabot, B; Blanchette, M; Lapierre, I; La Branche, H

1997-01-01

47

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing  

PubMed Central

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1–HIND interaction, cannot use certain non-canonical 5? splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.

Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M.; Krajewski, Marcin; Nagel, Roland J.; Ares, Manuel; Holak, Tad A.; Jentsch, Stefan

2013-01-01

48

Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron.  

PubMed Central

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point.

Kennedy, C F; Berget, S M

1997-01-01

49

Genomic splice site prediction algorithm based on nucleotide sequence pattern for RNA viruses  

Microsoft Academic Search

Splice site prediction on an RNA virus has two potential difficulties seriously degrading the performance of most conventional splice site predictors. One is a limited number of strains available for a virus species and the other is the diversified sequence patterns around the splice sites caused by the high mutation frequency. To overcome these two difficulties, a new algorithm called

Kun-Nan Tsai; Shu-Hung Lin; Shin-Ru Shih; Jhih-Siang Lai; Chung-Ming Chen

2009-01-01

50

Preventing seroma in the latissimus dorsi flap donor site  

Microsoft Academic Search

A technique for preventing seroma in latissimus dorsi flap donor sites is presented. The procedure involves quilting the donor site skin flaps to the underlying tissues with absorbable sutures.In a single surgeon series, a retrospective group (n=16) of non-quilted donor sites was compared to a prospective group of quilted donor sites (n =11). The age range and indications for surgery

O. G. Titley; G. E. Spyrou; M. F. T. Fatah

1997-01-01

51

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

PubMed Central

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.

Sowden, M P; Smith, H C

2001-01-01

52

Analysis by illegitimate transcription of a mutation in the 5{prime} splice site in exon 8 of the PAH gene  

SciTech Connect

Up to now, 12 splice defects have been described within the PAH gene. Using PCR-SSCP and sequence analysis we have found a point mutation involving the last nucleotide in exon 8 (CAG/CAA). The G to A substitution does not alter the amino acid (Q204Q), but it may cause a splice defect, as it is included in the 5{prime} splice donor site, and the G at this position is highly conserved (80%) in all eukaryotic genes. We have analyzed by illegitimate transcription the PAH mRNA in lymphocytes of a patient bearing the mutation in a heterozygous fashion. After RT-PCR we observed once the appearance of an extra larger band, which could be due to the use of a cryptic splice site instead of the mutated one. Furthermore, sequencing of 6 clones of the band of expected size in the patient revealed that all had the normal sequence, in spite of the G to A substitution being found in the genomic DNA. In view of these results, we believe that the larger extra band represents the allele with the mutation which causes a highly unstable mis-spliced RNA. This splice defect could be, therefore, the disease causing mutation in the patient.

Desviat, L.R.; Perez, B.; Ugarte, M. [CSICUAM, Cantoblanco, Madrid (Spain)

1994-09-01

53

Activation of cryptic splice sites in murine sarcoma virus-124 mutants.  

PubMed Central

We have examined splice site activation in relation to intron structure in murine sarcoma virus (MuSV)-124 RNA. MuSV-124 contains inactive murine leukemia virus env gene splice sites (termed 5' env and 3' env) as well as cryptic sites in the gag and v-mos genes (termed 5' gag and 3' mos) which are activated for thermosensitive splicing by a 1,487-base intronic deletion in the MuSV-124 derived MuSVts110 retrovirus. To determine conditions permissive for splice site activation, we examined MuSV-124 mutants deleted in the 1,919-base intron bounded by the 5' gag and 3' mos sites. Several of these deletions activated thermosensitive splicing either at the same sites used in MuSVts110 or in a previously unreported temperature-sensitive splice event between the 5' gag and 3' env sites. These data suggested that the thermosensitive splicing phenotype characteristic of MuSVts110 required neither a specialized intron nor selection of a particular 3' splice site. The 3' env and 3' mos sites were found to compete for splicing to the 5' gag site; the more upstream 3' env site was exclusively used in MuSV-124 mutants containing both sites, whereas selection of the 3' mos site required removal of the 3' env site. Branchpoint sequences were found to have a potential regulatory role in thermosensitive splicing. Insertion of a beta-globin branchpoint sequence in a splicing-inactive MuSV-124 mutant activated efficient nonthermosensitive splicing at the 3' mos site, whereas a mutated branchpoint activated less efficient but thermosensitive splicing. Images

de Mars, M; Cizdziel, P E; Murphy, E C

1990-01-01

54

Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus  

PubMed Central

Background Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus. Results By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants. Conclusion We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.

S?rensen, Annette Balle; Lund, Anders H; Kunder, Sandra; Quintanilla-Martinez, Leticia; Schmidt, Jorg; Wang, Bruce; Wabl, Matthias; Pedersen, Finn Skou

2007-01-01

55

Negative pressure wound dressing of the radial forearm donor site  

Microsoft Academic Search

Donor site complications of the radial forearm are a significant cause of post-operative morbidity. 15 patients had radial forearm free tissue donor sites treated with split skin grafts and a negative pressure dressing. All grafts showed 100% take at 5 days. The advantages of this technique include rapid healing at an unfavourable graft recipient site, increased graft take and decreased

C. Avery; J. Pereira; A. Moody; M. Gargiulo; I. Whitworth

2000-01-01

56

Human Splice Site Identification with Multiclass Support Vector Machines and Bagging  

Microsoft Academic Search

The complete identification of human genes involves determining parts that generates proteins, named exons, and those that do not code for proteins, known as introns. The splice site identification problem is concerned with the recognition of the boundaries between these regions. This work\\u000a investigates the use of Support Vector Machines (SVMs) in human splice site identification. Two methods employed for

Ana Carolina Lorena; André Carlos Ponce Leon Ferreira De Carvalho

2003-01-01

57

Evidence that RNA editing modulates splice site selection in the 5-HT2C receptor gene  

PubMed Central

Adenosine to inosine editing of mRNA from the human 5-HT2C receptor gene (HTR2C) occurs at five exonic positions (A–E) in a stable stem–loop that includes the normal 5? splice site of intron 5 and is flanked by two alternative splice sites. Using in vitro editing, we identified a novel editing site (F) located in the intronic part of the stem–loop and demonstrated editing at this site in human brain. We have shown that in cell culture, base substitutions to mimic editing at different combinations of the six sites profoundly affect relative splicing at the normal and the upstream alternative splice site, but splicing at the downstream alternative splice site was consistently rare. Editing combinations in different splice variants from human brain were determined and are consistent with the effects of editing on splicing observed in cell culture. As RNA editing usually occurs close to exon/intron boundaries, this is likely to be a general phenomenon and suggests an important novel role for RNA editing.

Flomen, Rachel; Knight, Joanne; Sham, Pak; Kerwin, Robert; Makoff, Andrew

2004-01-01

58

The spliced leader sequence of Trypanosoma brucei has a potential role as a cap donor structure.  

PubMed Central

Trypanosoma brucei brucei and other trypanosomatid species are unique among eucaryotes because transcription of their protein-coding genes is discontinuous. The 5' ends of their mRNAs consist of an identical 35-nucleotide spliced leader which is encoded at a separate locus from that for the body of the protein-coding transcript. We show here that the spliced leader transcript contains a 5' cap structure and suggest that at least one function of the spliced leader sequence is to provide a cap structure to trypanosome mRNAs. Images

Lenardo, M J; Dorfman, D M; Donelson, J E

1985-01-01

59

A novel splice-site mutation of TULP1 underlies severe early-onset retinitis pigmentosa in a consanguineous Israeli Muslim Arab family  

PubMed Central

Purpose To investigate the genetic basis for autosomal recessive severe early-onset retinitis pigmentosa (RP) in a consanguineous Israeli Muslim Arab family. Methods Haplotype analysis for all known genes underlying autosomal recessive RP was performed. Mutation screening of the underlying gene was done by direct sequencing. An in vitro splicing assay was used to evaluate the effect of the identified mutation on splicing. Results Haplotype analysis indicated linkage to the Tubby-like protein 1 (TULP)1 gene. Direct sequencing revealed a homozygous single base insertion, c.1495+2_1495+3insT, located in the conserved donor splice-site of intron 14. This mutation co-segregated with the disease, and was not detected in 114 unrelated Israeli Muslim Arab controls. We used an in vitro splicing assay to demonstrate that this mutation leads to incorrect splicing. Conclusions To date, 22 distinct pathogenic mutations of TULP1 have been reported in patients with early-onset RP or Leber congenital amaurosis. Here we report a novel splice-site mutation of TULP1, c.1495+2_1495+3insT, underlying autosomal recessive early-onset RP in a consanguineous Israeli Muslim Arab family. This report expands the spectrum of pathogenic mutations of the TULP1 gene.

Abbasi, Anan H.; Garzozi, Hanna J.

2008-01-01

60

Two novel mutations affecting the same splice site of PKD1 correlate with different phenotypes in ADPKD.  

PubMed

Abstract Genetic heterogeneity is the main factor for significant variation in the course of autosomal dominant polycystic kidney disease (ADPKD). PKD1 patients have more severe renal outcomes compared with PKD2 patients. Co-inheritance of a mutation in both genes is associated with more severe phenotypes than that found with either mutation alone. However, the genotype-phenotype relationship is far from clear in ADPKD. Here, we observed two novel mutations, PKD1:c.12444G?>?A and PKD1:c.12444?+?1G?>?A, which alter the same splice donor site of intron 45, correlate with different renal outcomes. To explain the phenomenon, we analyzed the genic and allelic background of the patients, as well as the genetic modifiers, DKK3 and HNF-1? as suggested. Only PKD1 variants were found, which highlights the allelic influence of PKD1 gene to be the last candidate factor. Segregation analysis, online mutation prediction, and recurrence mutation searching were applied to sort the variants. However, none of variants was found to be damaging or associated with the disease except PKD1:c.12444G?>?A and PKD1:c.12444?+?1G?>?A. Cloning and sequencing of the mutated cDNA sequences had shown unexpected different splicing effects caused by the mutations. PKD1:c.12444?+?1G?>?A definitely destroyed the native splice site and created a novel donor site with truncating effect on PC1. In contrast, PKD1:c.12444G?>?A mainly weakened the site and decreased the expression of normal PC1. Since PC1 negatively regulates cell proliferation in the process of cyst formation and enlargement, our observation may explain this new genotype-phenotype correlation and help to improve genetic counseling and diagnosis of the disease. PMID:24575920

Yu, Chaowen; Li, Jing; Yuan, Zhaojian; Liu, Shan; Zou, Lin

2014-06-01

61

Activation of a cryptic 5' splice site by U1 snRNA.  

PubMed Central

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.

Alvarez, C J; Wise, J A

2001-01-01

62

Analysis by illegitimate transcription of a mutation in the 5â² splice site in exon 8 of the PAH gene  

Microsoft Academic Search

Up to now, 12 splice defects have been described within the PAH gene. Using PCR-SSCP and sequence analysis we have found a point mutation involving the last nucleotide in exon 8 (CAG\\/CAA). The G to A substitution does not alter the amino acid (Q204Q), but it may cause a splice defect, as it is included in the 5â² splice donor

L. R. Desviat; B. Perez; M. Ugarte

1994-01-01

63

The A1 and A1B proteins of heterogeneous nuclear ribonucleoparticles modulate 5' splice site selection in vivo.  

PubMed Central

Recent in vitro results suggest that the heterogeneous nuclear ribonucleoparticle (hnRNP) A1 protein modulates alternative splicing by favoring distal 5' splice site (5'SS) selection and exon skipping. We used a mouse erythroleukemia (MEL) cell line (CB3C7) deficient in the expression of hnRNP A1 to test whether variations in hnRNP A1 and AlB protein levels affected alternative splicing in vivo. In contrast to A1-expressing MEL cell lines, CB3C7 cells preferentially selected the proximal 13S and 12S 5'SS on the adenovirus E1A pre-mRNA. Transiently expressing the A1 or A1B cDNA in CB3C7 cells shifted 5'SS selection toward the more distal 9S donor site. A1 protein synthesis was required for this effect since the expression of a mutated A1 cDNA did not affect 5'SS selection. These results demonstrate that in vivo variations in hnRNP A1 protein levels can influence 5'SS selection. Images

Yang, X; Bani, M R; Lu, S J; Rowan, S; Ben-David, Y; Chabot, B

1994-01-01

64

Beta-hexosaminidase splice site mutation has a high frequency among non-Jewish Tay-Sachs disease carriers from the British Isles  

Microsoft Academic Search

In the course of defining mutations causing Tay-Sachs disease (TSD) in non-Jewish patients and carriers from the British Isles, we identified a guanine to adenine change (also previously described) in the obligatory GT sequence of the donor splice site at the 5' end of intron 9 of the hexosaminidase alpha peptide gene. Of 24 unrelated mutant chromosomes from 20 non-Jewish

E C Landels; P M Green; I H Ellis; A H Fensom; M Bobrow

1992-01-01

65

A competitive regulatory mechanism discriminates between juxtaposed splice sites and pri-miRNA structures  

PubMed Central

We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3? splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3?ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3? SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.

Mattioli, Chiara; Pianigiani, Giulia; Pagani, Franco

2013-01-01

66

Allele-specific recognition of the 3? splice site of INS intron 1  

PubMed Central

Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin gene (INS) and with differential steady-state levels of INS RNA from T1D-predisposing and -protective haplotypes. Here, we show that the haplotype-specific expression is determined by INS variants that control the splicing efficiency of intron 1. The adenine allele at IVS1-6 (rs689), which rapidly expanded in modern humans, renders the 3? splice site of this intron more dependent on the auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF). This interaction required both zinc fingers of the 35-kD U2AF subunit (U2AF35) and was associated with repression of a competing 3? splice site in INS exon 2. Systematic mutagenesis of reporter constructs showed that intron 1 removal was facilitated by conserved guanosine-rich enhancers and identified additional splicing regulatory motifs in exon 2. Sequencing of intron 1 in primates revealed that relaxation of its 3? splice site in Hominidae coevolved with the introduction of a short upstream open reading frame, providing a more efficient coupled splicing and translation control. Depletion of SR proteins 9G8 and transformer-2 by RNA interference was associated with exon 2 skipping whereas depletion of SRp20 with increased representation of transcripts containing a cryptic 3? splice site in the last exon. Together, these findings reveal critical interactions underlying the allele-dependent INS expression and INS-mediated risk of T1D and suggest that the increased requirement for U2AF35 in higher primates may hinder thymic presentation of autoantigens encoded by transcripts with weak 3? splice sites. Electronic supplementary material The online version of this article (doi:10.1007/s00439-010-0860-1) contains supplementary material, which is available to authorized users.

Kralovicova, Jana

2010-01-01

67

Bimolecular exon ligation by the human spliceosome bypasses early 3' splice site AG recognition and requires NTP hydrolysis.  

PubMed Central

Here we report further characterization of an in vitro assay system for exon ligation by the human spliceosome in which the 3' splice site AG is supplied by a different RNA molecule than that containing the 5' splice and branch sites. By varying the time during splicing reactions when the 3' splice site AG is made available to the splicing machinery, we show that AG recognition need not occur until after lariat formation. Thus an early AG recognition event required for spliceosome formation and lariat formation on some mammalian introns is not required for exon ligation. Depletion/add-back studies and cold competitor challenge experiments reveal that commitment of a 3' splice site AG to exon ligation requires NTP hydrolysis. Because it both physically and kinetically uncouples exon ligation from spliceosome assembly and lariat formation, the bimolecular system will be a valuable tool for further mechanistic analysis of the second step of splicing.

Anderson, K; Moore, M J

2000-01-01

68

Hepatitis B Virus DNA Splicing in Lebanese Blood Donors and Genotype A to E Strains: Implications for Hepatitis B Virus DNA Quantification and Infectivity  

PubMed Central

Hepatitis B virus (HBV) is one of the major viruses transmissible by blood that causes chronic infection in immunocompromised individuals. The study of 61 HBV carrier blood donors from Lebanon revealed multiple patterns of spliced HBV DNA. HBV DNA splicing was examined and quantified in samples of five genotypes and in seroconversion panels. The Lebanese sample median viral load was 1.5 ×102 IU/ml. All strains were genotype D, serotype ayw; 35 clustered as subgenotype D1 and 7 clustered as subgenotype D2. Three splice variants (SP1, SP1A, and Pol/S) were observed in 12 high-viral-load samples. Twenty samples of each genotype, A to E, were tested for the presence of HBV spliced DNA and SP1-specific splice variant. An unspliced HBV genome was dominant, but 100% of strains with a viral load of ?105 copies/ml contained various proportions of spliced DNA. SP1 was detected in 56/100 (56%) samples in levels that correlated with the overall viral load. HBV DNA quantification with S (unspliced) and X (total DNA) regions provided different levels of viral load, with the difference corresponding to spliced DNA. During the highly infectious window period, the SP1 variant became detectable shortly after the hepatitis B surface antigen (HBsAg), suggesting a correlation between the initiation of splicing and the production of detectable levels of HBsAg. The quantification of HBV DNA with primers located outside and inside the spliced region might provide different estimations of viral load and differentiate between infectious and defective viral genomes. The role of splicing neoproteins in HBV replication and interaction with the host remains to be determined.

El Chaar, Mira; El Jisr, Tamima

2012-01-01

69

Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations  

Microsoft Academic Search

The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat

Thilo Dörk; Rainer Fislage; Thomas Neumann; Brigitte Wulf; Burkhard Tümmler

1994-01-01

70

Factor interactions with the simian virus 40 early pre-mRNA influence branch site selection and alternative splicing.  

PubMed

To study the interaction of splicing factors with the simian virus 40 early-region pre-RNA, which can be alternatively spliced to produce large T and small t mRNAs, we used an in vitro RNase protection assay that defines the 5' boundaries of factor-RNA interactions. Protection products reflecting factor interactions with the large T and small t 5' splice sites and with the multiple lariat branch site region were characterized. All protection products were detected very early in the splicing reaction, before the appearance of spliced RNAs. However, protection of the large T 5' splice site was detected well before small t 5' splice site and branch site protection products, which appeared simultaneously. Oligonucleotide-targeted degradation of small nuclear RNAs (snRNAs) revealed that protection of the branch site region, which occurred at multiple sites, required intact U2 snRNA and was enhanced by U1 snRNA, while protection of the large T and small t 5' splice sites required both U1 and U2 snRNAs. Analysis of several pre-RNAs containing mutations in the branch site region suggests that factor interactions involving the multiple copies of the branch site consensus determine the selection of branch points, which is an important factor in the selection of alternative splicing pathways. PMID:2546057

Noble, J C; Ge, H; Chaudhuri, M; Manley, J L

1989-05-01

71

Virus deletion mutants that affect a 3' splice site in the E3 transcription unit of adenovirus 2.  

PubMed Central

Five viable virus mutants were constructed with deletions near a 3' splice site located at nucleotide 2157 in the E3 transcription unit of adenovirus 2. The mutants were examined for splicing activity at the 2157 3' splice site in vivo by nuclease-gel analysis of steady-state cytoplasmic mRNA. Splicing was not prevented by an exon deletion (dl719) that leaves 16 5'-proximal exon nucleotides intact or by intron deletions that leave 34 (dl717, dl712) or 18 (dl716) 3'-proximal intron nucleotides intact. The sequences deleted in one of these intron mutants (dl716) include the putative branchpoint site used in lariat formation during splicing. Thus, a surrogate branchpoint site apparently can be used for splicing. Another intron mutant (dl714) has a deletion that leaves 15 3'-proximal intron nucleotides intact; remarkably, this deletion virtually abolished splicing, even though the deletion is only 3 nucleotides closer to the splice site than is the deletion in dl716 which splices normally. The three nucleotides deleted in dl714 that are retained by dl716 are the sequence TGT. The TGT sequence is located on the 5' boundary of the pyrimidine-rich region upstream of the nucleotide 2157 3' splice site. Such pyrimidine-rich regions are ubiquitous at 3' splice sites. Most likely, the TGT is required for splicing at the nucleotide 2157 3' splice site. The TGT may be important because of its specific sequence or because it forms the 5' boundary of the pyrimidine-rich region. Images

Bhat, B M; Brady, H A; Wold, W S

1985-01-01

72

Beta-hexosaminidase splice site mutation has a high frequency among non-Jewish Tay-Sachs disease carriers from the British Isles.  

PubMed Central

In the course of defining mutations causing Tay-Sachs disease (TSD) in non-Jewish patients and carriers from the British Isles, we identified a guanine to adenine change (also previously described) in the obligatory GT sequence of the donor splice site at the 5' end of intron 9 of the hexosaminidase alpha peptide gene. Of 24 unrelated mutant chromosomes from 20 non-Jewish subjects (15 TSD carriers, four TSD patients, and one TSD fetus), five had mutations common in the Ashkenazi Jewish community, and 10 had the intron 9 splice site mutation. This is an unexpected result considering the diverse origin of the population of the British Isles. This mutation was not found in 28 control UK subjects or 11 Jewish carriers of known TSD mutations. Before attempting detection of unknown mutations, non-Jewish TSD carriers from the British Isles should be screened for the intron 9 donor splice site mutation as well as those mutations which predominate in the Jewish community. Images

Landels, E C; Green, P M; Ellis, I H; Fensom, A H; Bobrow, M

1992-01-01

73

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5? splice site and branch site  

PubMed Central

Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5? splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.

Crawford, Daniel J.; Hoskins, Aaron A.; Friedman, Larry J.; Gelles, Jeff; Moore, Melissa J.

2013-01-01

74

DNA splice site detection: a comparison of specific and general methods.  

PubMed

In an era when whole organism genomes are being routinely sequenced, the problem of gene finding has become a key issue on the road to understanding. For eukaryotic organisms a large part of locating the genes is accomplished by predicting the likely location of splice sites on a DNA strand. This problem of splice site location has been ap- proached using a number of machine learning or statistical methods tailored more or less specifically to the nature of the problem. Recently large margin classifiers and boosting methods have been found to give improvements over more traditional methods in a number of areas. Here we compare large margin classifiers (SVM and CMLS) and boosted decision trees with the three most common models used for splice site detection (WMM, WAM, and MDT). We find that the newer methods compare favorably in all cases and can yield significant improvement in some cases. PMID:12463853

Kim, Won; Wilbur, W John

2002-01-01

75

Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing  

PubMed Central

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.

Farrer, Tracy; Roller, A. Brock; Kent, W. James; Zahler, Alan M.

2002-01-01

76

Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance.  

PubMed Central

One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both. Images

Cohen, J B; Snow, J E; Spencer, S D; Levinson, A D

1994-01-01

77

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome.  

PubMed

Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families. PMID:23592335

Rice, Gillian I; Reijns, Martin A M; Coffin, Stephanie R; Forte, Gabriella M A; Anderson, Beverley H; Szynkiewicz, Marcin; Gornall, Hannah; Gent, David; Leitch, Andrea; Botella, Maria P; Fazzi, Elisa; Gener, Blanca; Lagae, Lieven; Olivieri, Ivana; Orcesi, Simona; Swoboda, Kathryn J; Perrino, Fred W; Jackson, Andrew P; Crow, Yanick J

2013-08-01

78

Pick one, but be quick: 5' splice sites and the problems of too many choices.  

PubMed

Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 5' Splice sites (5'ss) are the critical elements at the 5' end of introns and are extremely diverse, as thousands of different sequences act as bona fide 5'ss in the human transcriptome. Most 5'ss are recognized by base-pairing with the 5' end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 5'ss selection, highlighting the difficulties of establishing how base-pairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:5'ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 5'ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 5'ss might lead to selection of a single 5'ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 5'ss selection. PMID:23348838

Roca, Xavier; Krainer, Adrian R; Eperon, Ian C

2013-01-15

79

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

SciTech Connect

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.

Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. (National Institutes of Health, Bethesda, MD (United States) Erasmus Univ. of Rotterdam (Netherlands))

1993-03-01

80

A novel splice site mutation of CDHR1 in a consanguineous Israeli Christian Arab family segregating autosomal recessive cone-rod dystrophy  

PubMed Central

Purpose To investigate the genetic basis for autosomal recessive cone-rod dystrophy in a consanguineous Israeli Christian Arab family. Methods Patients underwent a detailed ophthalmic examination, including funduscopy, electroretinography (ERG), visual field testing, and optical coherence tomography. Genome-wide homozygosity mapping using a single nucleotide polymorphism array was performed to identify homozygous regions shared between the two affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico analysis was used to predict the effect of the mutation on splicing. Results The family included two affected individuals. Clinical findings included progressive deterioration of visual acuity, photophobia, defective color vision, loss of central visual fields, pigmentary deposits localized mainly in the peripheral retina, a thinned and atrophic macular region, retinal vessel attenuation, absent ERG cone responses, and reduced ERG rod responses. Homozygosity mapping revealed several homozygous intervals shared among the affected individuals. One, a 12Mb interval on chromosome 10, included the CDHR1 gene. Direct sequencing revealed a single base transversion, c.1485+2T>G, located in the conserved donor splice site of Intron 13. This mutation cosegregated with the disease in the family, and was not detected in 208 Israeli Christian Arab control chromosomes. In silico analysis predicted that this mutation eliminates the Intron 13 donor splice site. Conclusions Only three distinct pathogenic mutations of CDHR1 have been reported to date in patients with autosomal recessive retinal degeneration. Here we report a novel splice site mutation of CDHR1, c.1485+2T>G, underlying autosomal recessive cone-rod dystrophy in a consanguineous Israeli Christian Arab family. This report expands the spectrum of pathogenic mutations of the CDHR1 gene.

Cohen, Ben; Chervinsky, Elena; Jabaly-Habib, Haneen; Shalev, Stavit A.; Briscoe, Daniel

2012-01-01

81

Somatic mosaicism for a newly identified splice-site mutation in a patient with adenosine deaminase-deficient immunodeficiency and spontaneous clinical recovery  

SciTech Connect

Absent or severely reduced adenosine deaminase (ADA) activity produces inherited immunodeficiency of varying severity, with defects of both cellular and humoral immunity. The authors report somatic mosaicism as the basis for a delayed presentation and unusual course of a currently healthy young adult receiving no therapy. He was diagnosed at age 2[1/2] years because of life-threatening pneumonia, recurrent infections, failure of normal growth, and lymphopenia, but he retained significant cellular immune function. A fibroblast cell line and a B cell line, established at diagnosis, lacked ADA activity and were heteroallelic for a splice-donor-site mutation in IVS 1 (+1GT[yields]CT) and a missense mutation (Arg101Gln). All clones (17/17) isolated from the B cell mRNA carried the missense mutation, indicating that the allele with the splice-site mutation produced unstable mRNA. In striking contrast, a B cell line established at age 16 years expressed 50% of normal ADA; 50% had the missense mutation. Genomic DNA contained the missense mutation but not the splice-site mutation. All three cell lines were identical for multiple polymorphic markers and the presence of a Y chromosome. In vivo somatic mosaicism was demonstrated in genomic DNA from peripheral blood cells obtained at 16 years of age, in that less than half the DNA carried the splice-site mutation (P<.0.02, vs. original B cell line). Consistent with mosaicism, erythrocyte content of the toxic metabolite deoxyATP was only minimally elevated. Somatic mosaicism could have arisen either by somatic mutation or by reversion at the site of mutation. Selection in vivo for ADA normal hematopoietic cells may have played a role in the return to normal health, in the absence of therapy. 57 refs., 4 figs., 2 tabs.

Hirschhorn, R.; Yang, D.R.; Israni, A.; Huie, M.L. (New York Univ. Medical Center, NY (United States)); Ownby, D.R. (Henry Ford Hospital, Detroit, MI (United States))

1994-07-01

82

The transition in spliceosome assembly from complex E to complex A purges surplus U1 snRNPs from alternative splice sites  

PubMed Central

Spliceosomes are assembled in stages. The first stage forms complex E, which is characterized by the presence of U1 snRNPs base-paired to the 5? splice site, components recognizing the 3? splice site and proteins thought to connect them. The splice sites are held in close proximity and the pre-mRNA is committed to splicing. Despite this, the sites for splicing appear not to be fixed until the next complex (A) forms. We have investigated the reasons why 5? splice sites are not fixed in complex E, using single molecule methods to determine the stoichiometry of U1 snRNPs bound to pre-mRNA with one or two strong 5? splice sites. In complex E most transcripts with two alternative 5? splice sites were bound by two U1 snRNPs. However, the surplus U1 snRNPs were displaced during complex A formation in an ATP-dependent process requiring an intact 3? splice site. This process leaves only one U1 snRNP per complex A, regardless of the number of potential sites. We propose a mechanism for selection of the 5? splice site. Our results show that constitutive splicing components need not be present in a fixed stoichiometry in a splicing complex.

Hodson, Mark J.; Hudson, Andrew J.; Cherny, Dmitry; Eperon, Ian C.

2012-01-01

83

The transition in spliceosome assembly from complex E to complex A purges surplus U1 snRNPs from alternative splice sites.  

PubMed

Spliceosomes are assembled in stages. The first stage forms complex E, which is characterized by the presence of U1 snRNPs base-paired to the 5' splice site, components recognizing the 3' splice site and proteins thought to connect them. The splice sites are held in close proximity and the pre-mRNA is committed to splicing. Despite this, the sites for splicing appear not to be fixed until the next complex (A) forms. We have investigated the reasons why 5' splice sites are not fixed in complex E, using single molecule methods to determine the stoichiometry of U1 snRNPs bound to pre-mRNA with one or two strong 5' splice sites. In complex E most transcripts with two alternative 5' splice sites were bound by two U1 snRNPs. However, the surplus U1 snRNPs were displaced during complex A formation in an ATP-dependent process requiring an intact 3' splice site. This process leaves only one U1 snRNP per complex A, regardless of the number of potential sites. We propose a mechanism for selection of the 5' splice site. Our results show that constitutive splicing components need not be present in a fixed stoichiometry in a splicing complex. PMID:22505580

Hodson, Mark J; Hudson, Andrew J; Cherny, Dmitry; Eperon, Ian C

2012-08-01

84

Alternative splicing tends to avoid partial removals of protein-protein interaction sites  

PubMed Central

Background Anecdotal evidence of the involvement of alternative splicing (AS) in the regulation of protein-protein interactions has been reported by several studies. AS events have been shown to significantly occur in regions where a protein interaction domain or a short linear motif is present. Several AS variants show partial or complete loss of interface residues, suggesting that AS can play a major role in the interaction regulation by selectively targeting the protein binding sites. In the present study we performed a statistical analysis of the alternative splicing of a non-redundant dataset of human protein-protein interfaces known at molecular level to determine the importance of this way of modulation of protein-protein interactions through AS. Results Using a Cochran-Mantel-Haenszel chi-square test we demonstrated that the alternative splicing-mediated partial removal of both heterodimeric and homodimeric binding sites occurs at lower frequencies than expected, and this holds true even if we consider only those isoforms whose sequence is less different from that of the canonical protein and which therefore allow to selectively regulate functional regions of the protein. On the other hand, large removals of the binding site are not significantly prevented, possibly because they are associated to drastic structural changes of the protein. The observed protection of the binding sites from AS is not preferentially directed towards putative hot spot interface residues, and is widespread to all protein functional classes. Conclusions Our findings indicate that protein-protein binding sites are generally protected from alternative splicing-mediated partial removals. However, some cases in which the binding site is selectively removed exist, and here we discuss one of them.

2013-01-01

85

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

PubMed Central

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.

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

1999-01-01

86

Occlusive versus semi-open dressings in the management of skin graft donor sites.  

PubMed

In a prospective, randomised study we compared the efficacy of a new occlusive dressing (Granuflex-E) with a semi-open dressing (tulle gras) in the management of skin-graft donor sites. The study examined 10 patients with burns, each with two donor sites. Each patient acted as his or her own control. The donor sites were mirror images of each other, the anterior surface of the thighs. One donor site was dressed with Granuflex-E and the other with tulle gras. The sites dressed with the occlusive dressing healed significantly faster and were more comfortable than the sites with the semi-open dressing. PMID:1519121

Demetriades, D; Psaras, G

1992-06-01

87

De novo SCN2A splice site mutation in a boy with Autism spectrum disorder  

PubMed Central

Background SCN2A is a gene that codes for the alpha subunit of voltage-gated, type II sodium channels, and is highly expressed in the brain. Sodium channel disruptions, such as mutations in SCN2A, may play an important role in psychiatric disorders. Recently, de novo SCN2A mutations in autism spectrum disorder (ASD) have been identified. The current study characterizes a de novo splice site mutation in SCN2A that alters mRNA and protein products. Case presentation We describe results from clinical and genetic characterizations of a seven-year-old boy with ASD. Psychiatric interview and gold standard autism diagnostic instruments (ADOS and ADI-R) were used to confirm ASD diagnosis, in addition to performing standardized cognitive and adaptive functioning assessments (Leiter-R and Vineland Adaptive Behavior Scale), and sensory reactivity assessments (Sensory Profile and Sensory Processing Scales). Genetic testing by whole exome sequencing revealed four de novo events, including a splice site mutation c.476?+?1G?>?A in SCN2A, a missense mutation (c.2263G?>?A) causing a p.V755I change in the TLE1 gene, and two synonymous mutations (c.2943A?>?G in the BUB1 gene, and c.1254 T?>?A in C10orf68 gene). The de novo SCN2A splice site mutation produced a stop codon 10 amino acids downstream, possibly resulting in a truncated protein and/or a nonsense-mediated mRNA decay. The participant met new DSM-5 criteria for ASD, presenting with social and communication impairment, repetitive behaviors, and sensory reactivity issues. The participant’s adaptive and cognitive skills fell in the low range of functioning. Conclusion This report indicates that a splice site mutation in SCN2A might be contributing to the risk of ASD. Describing the specific phenotype associated with SCN2A mutations might help to reduce heterogeneity seen in ASD.

2014-01-01

88

A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment  

PubMed Central

The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects.

Gandia, Marta; del Castillo, Francisco J.; Rodriguez-Alvarez, Francisco J.; Garrido, Gema; Villamar, Manuela; Calderon, Manuela; Moreno-Pelayo, Miguel A.; Moreno, Felipe; del Castillo, Ignacio

2013-01-01

89

Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect  

Microsoft Academic Search

Hereditary forms of hearing impairment (HI) caused by GJB2 (Cx26) mutations are the frequent sensory disorders registered among newborns in various human populations. In this study, we present data on the molecular, audiological and population features of autosomal recessive deafness 1A (DFNB1A) associated with the donor splicing site IVS1+1G>A mutation of GJB2 gene in Yakut population isolate of the Sakha

Nikolay A Barashkov; Lilya U Dzhemileva; Sardana A Fedorova; Fedor M Teryutin; Olga L Posukh; Elvira E Fedotova; Simeon L Lobov; Elza K Khusnutdinova

2011-01-01

90

The nematode spliced leader RNA participates in trans-splicing as an Sm snRNP.  

PubMed

The trans-spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans-splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans-splicing. The effect on trans-splicing is not due to undermethylation of the cap structure. PMID:2145151

Maroney, P A; Hannon, G J; Denker, J A; Nilsen, T W

1990-11-01

91

The nematode spliced leader RNA participates in trans-splicing as an Sm snRNP.  

PubMed Central

The trans-spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans-splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans-splicing. The effect on trans-splicing is not due to undermethylation of the cap structure. Images Fig.1 Fig.2 Fig.3 Fig.4 Fig.5 Fig.6

Maroney, P A; Hannon, G J; Denker, J A; Nilsen, T W

1990-01-01

92

Spontaneous deletions in Ig heavy chain genes: flanking sequences influence splice site selection.  

PubMed Central

The cell line G403.4.7, isolated as a spontaneous variant of the MPC-11 derived myeloma G403.4, produces a truncated gamma 2b HC protein, but no light chain (LC), and a single gamma 2b specific transcript of 2.4kb. This gamma 2b transcript consists of the VDJ and CH1 exons, the CH1 to Hinge (Hi) intervening sequence (IVS) and HI exon, part of the IVS between the two membrane exons M1 and M2, and most of the membrane 3' untranslated (UT) region. Even though the mature mRNA contains intronic sequences, it is abundant in the cytoplasm. Analysis of the gamma 2b genomic organization reveals that this unusual transcript results in part from two genomic deletions of 2.5kb and 588bp and in part from an altered splicing pattern. This altered splicing pattern is probably a consequence of the sequence alterations resulting from the genomic deletions. Analysis of these events provides some interesting insights into the mechanism of splice site selection and the evolution of introns and exons. Images

Ward, S B; Morrison, S L

1991-01-01

93

Biased exon/intron distribution of cryptic and de novo 3? splice sites  

PubMed Central

We compiled sequences of previously published aberrant 3? splice sites (3?ss) that were generated by mutations in human disease genes. Cryptic 3?ss, defined here as those resulting from a mutation of the 3?YAG consensus, were more frequent in exons than in introns. They clustered in ?20 nt region adjacent to authentic 3?ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3?ss that were induced by mutations outside the 3?YAG consensus (designated ‘de novo’) were in introns. The activation of intronic de novo 3?ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3?ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro–Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3?ss. Finally, AG-creating mutations in the PPT that produced aberrant 3?ss upstream of the predicted BPS in vivo shared a similar ‘BPS-new AG’ distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3?ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.

Kralovicova, Jana; Christensen, Mikkel B.; Vorechovsky, Igor

2005-01-01

94

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites.  

PubMed Central

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells. Images

Montell, C; Fisher, E F; Caruthers, M H; Berk, A J

1984-01-01

95

Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3? Splice Site In Vivo  

PubMed Central

The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the in vivo regulation of splicing. From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature-sensitive lethality and caused the unc-93 RNA transcript to be spliced using a cryptic 3? splice site generated by the unc-93(e1500) missense mutation. The sfa-1(n4562) mutation did not cause the utilization of this cryptic 3? splice site. We isolated four uaf-1(n4588) intragenic suppressors that restored the viability of uaf-1 mutants at 25°C. These suppressors differentially affected the recognition of the cryptic 3? splice site and implicated a small region of UAF-1 between the U2AF small subunit-interaction domain and the first RNA recognition motif in affecting the choice of 3? splice site. We constructed a reporter for unc-93 splicing and using site-directed mutagenesis found that the position of the cryptic splice site affects its recognition. We also identified nucleotides of the endogenous 3? splice site important for recognition by wild-type UAF-1. Our genetic and molecular analyses suggested that the phenotypic suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588) and sfa-1(n4562) was likely caused by altered splicing of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 3? splice-site choice and establish a system that can be used to investigate the in vivo regulation of RNA splicing in C. elegans.

Ma, Long; Horvitz, H. Robert

2009-01-01

96

Identification and characterization of a novel splice site mutation in the SERPING1 gene in a family with hereditary angioedema.  

PubMed

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal-dominant disease caused by mutations in SERPING1 gene. The main clinical feature of C1INH deficiency is the spontaneous edema of the subcutaneous and submucosal layers. More than 280 different mutations scattering the entire SERPING1 gene have been reported. We identified and characterized a new mutation in SERPING1 gene in a Spanish family with hereditary angioedema. The mutation (c.685 + 2 T > A) disrupts the donor splice site of intron 4 leading to the loss of exon 4 in mutant mRNA. We demonstrated that mutant mRNA is mostly degraded, probably by the surveillance pathway no-go mRNA decay. Bioinformatic analysis showed that the mutant protein, if produced, would be non-functional since the protein lacks a stretch of 45 amino acids affecting the functional RCL loop. Finally, we found a reduction of the wild-type mRNA expression in c.685 + 2 T > A carriers. PMID:24412907

Colobran, Roger; Lois, Sergio; de la Cruz, Xavier; Pujol-Borrell, Ricardo; Hernández-González, Manuel; Guilarte, Mar

2014-02-01

97

Colorimetric examination of typical free flap donor sites and comparison to recipient sites in the extremities.  

PubMed

Results after free flap reconstruction in the extremities are often impaired by missing color match of the transferred flap and the recipient site. But pre-existing color match is the precondition for satisfying aesthetic results. To obtain suitable free flap donor sites in terms of color for extremity reconstruction and to understand frequent color mismatch, we performed a colorimetric study including 60 healthy volunteers. Ten free flap donor sites were compared with ten recipient sites in the extremities. The results of our study showed that lower extremity sites are markedly lighter than upper extremity sites with the exception of the palmar forearm. We encountered an excellent color match of the radial forearm flap to the back of the hand (4.10 ± 1.91) and the palm of the hand (5.62 ± 2.21), and significantly relevant color match to the palmar aspect of the forearm (2.52 ± 1.23). Additionally, the lateral arm flap showed a remarkable color match to the dorsal aspect of the forearm (3.13 ± 2.06). Furthermore we encountered significantly relevant color match of the fibula flap to the anterior aspect of the lower leg (2.01 ± 1.08) and excellent color match of the anterolateral thigh flap (ALT) to the palmar aspect of the forearm (3.66 ± 2.10). No further significantly relevant color differences between the other donor sites and recipient regions were found. Colorimetric measurements are a helpful tool in reconstructive surgery to compare skin color of different anatomic sites. PMID:23093467

Ensat, Florian; Greindl, Markus; Skreiner, Anna; Schubert, Heinrich; Hladik, Michaela; Stuflesser, Alexander; Spies, Marcus; Wechselberger, Gottfried

2013-01-01

98

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

PubMed Central

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.

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

2001-01-01

99

Complete human gene structure of obscurin: implications for isoform generation by differential splicing  

Microsoft Academic Search

The complete gene giant muscle protein obscurin, a modular protein composed largely of tandem Ig-domains, GDP\\/GTP exchange factor domains (GEF) for small G-proteins, and differentially spliced kinase domains, was analysed. The splice donor and acceptor sites of the 117 exons give important clues for potential splice pathways. The fusion of the conventional obscurin A, containing only the GEF domain, and

Atsushi Fukuzawa; Seraphina Idowu; Mathias Gautel

2005-01-01

100

The CUGBP2 Splicing Factor Regulates an Ensemble of Branchpoints from Perimeter Binding Sites with Implications for Autoregulation  

PubMed Central

Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter–binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns.

Dembowski, Jill A.; Grabowski, Paula J.

2009-01-01

101

Weak Negative and Positive Selection and the Drift Load at Splice Sites  

PubMed Central

Splice sites (SSs) are short sequences that are crucial for proper mRNA splicing in eukaryotic cells, and therefore can be expected to be shaped by strong selection. Nevertheless, in mammals and in other intron-rich organisms, many of the SSs often involve nonconsensus (Nc), rather than consensus (Cn), nucleotides, and beyond the two critical nucleotides, the SSs are not perfectly conserved between species. Here, we compare the SS sequences between primates, and between Drosophila fruit flies, to reveal the pattern of selection acting at SSs. Cn-to-Nc substitutions are less frequent, and Nc-to-Cn substitutions are more frequent, than neutrally expected, indicating, respectively, negative and positive selection. This selection is relatively weak (1 < |4Nes| < 4), and has a similar efficiency in primates and in Drosophila. Within some nucleotide positions, the positive selection in favor of Nc-to-Cn substitutions is weaker than the negative selection maintaining already established Cn nucleotides; this difference is due to site-specific negative selection favoring current Nc nucleotides. In general, however, the strength of negative selection protecting the Cn alleles is similar in magnitude to the strength of positive selection favoring replacement of Nc alleles, as expected under the simple nearly neutral turnover. In summary, although a fraction of the Nc nucleotides within SSs is maintained by selection, the abundance of deleterious nucleotides in this class suggests a substantial genome-wide drift load.

Denisov, Stepan V.; Bazykin, Georgii A.; Sutormin, Roman; Favorov, Alexander V.; Mironov, Andrey A.; Gelfand, Mikhail S.; Kondrashov, Alexey S.

2014-01-01

102

Weak negative and positive selection and the drift load at splice sites.  

PubMed

Splice sites (SSs) are short sequences that are crucial for proper mRNA splicing in eukaryotic cells, and therefore can be expected to be shaped by strong selection. Nevertheless, in mammals and in other intron-rich organisms, many of the SSs often involve nonconsensus (Nc), rather than consensus (Cn), nucleotides, and beyond the two critical nucleotides, the SSs are not perfectly conserved between species. Here, we compare the SS sequences between primates, and between Drosophila fruit flies, to reveal the pattern of selection acting at SSs. Cn-to-Nc substitutions are less frequent, and Nc-to-Cn substitutions are more frequent, than neutrally expected, indicating, respectively, negative and positive selection. This selection is relatively weak (1 < |4Nes| < 4), and has a similar efficiency in primates and in Drosophila. Within some nucleotide positions, the positive selection in favor of Nc-to-Cn substitutions is weaker than the negative selection maintaining already established Cn nucleotides; this difference is due to site-specific negative selection favoring current Nc nucleotides. In general, however, the strength of negative selection protecting the Cn alleles is similar in magnitude to the strength of positive selection favoring replacement of Nc alleles, as expected under the simple nearly neutral turnover. In summary, although a fraction of the Nc nucleotides within SSs is maintained by selection, the abundance of deleterious nucleotides in this class suggests a substantial genome-wide drift load. PMID:24966225

Denisov, Stepan V; Bazykin, Georgii A; Sutormin, Roman; Favorov, Alexander V; Mironov, Andrey A; Gelfand, Mikhail S; Kondrashov, Alexey S

2014-01-01

103

Reverse splicing of the Tetrahymena IVS: evidence for multiple reaction sites in the 23S rRNA.  

PubMed Central

Group I introns in rRNA genes are clustered in highly conserved regions that include tRNA and mRNA binding sites. This pattern is consistent with insertion of group I introns by direct interaction with exposed regions of rRNA. Integration of the Tetrahymena group I intron (or intervening sequence, IVS) into large subunit rRNA via reverse splicing was investigated using E. coli 23S rRNA as a model substrate. The results show that sequences homologous to the splice junction in Tetrahymena are the preferred site of integration, but that many other sequences in the 23S rRNA provide secondary targets. Like the original splice junction, many new reaction sites are in regions of stable secondary structure. Reaction at the natural splice junction is observed in 50S subunits and to a lesser extent in 70S ribosomes. These results support the feasibility of intron transposition to new sites in rRNA genes via reverse splicing. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6 FIGURE 7

Roman, J; Woodson, S A

1995-01-01

104

Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3'-splice site recognition.  

PubMed

Recognition of the 3'-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein-protein and protein-RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1(NTD)). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1(NTD) with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65(UHM)) reveals that, in addition to the known U2AF65(UHM)-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65(UHM), which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1-U2AF65-RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1-U2AF65 or the SF1-U2AF65-RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3'-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1-U2AF65-RNA complex. PMID:23175611

Zhang, Yun; Madl, Tobias; Bagdiul, Ivona; Kern, Thomas; Kang, Hyun-Seo; Zou, Peijian; Mäusbacher, Nina; Sieber, Stephan A; Krämer, Angela; Sattler, Michael

2013-01-01

105

Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3?-splice site recognition  

PubMed Central

Recognition of the 3?-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65UHM) reveals that, in addition to the known U2AF65UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3?-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA complex.

Zhang, Yun; Madl, Tobias; Bagdiul, Ivona; Kern, Thomas; Kang, Hyun-Seo; Zou, Peijian; Mausbacher, Nina; Sieber, Stephan A.; Kramer, Angela; Sattler, Michael

2013-01-01

106

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

SciTech Connect

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.

Hunter, T.C.; Albertini, R.J.; O`Neill, J.P. [Univ. of Vermont Genetics Lab., Burlington, VT (United States)] [and others

1996-03-01

107

Squamous cell carcinoma of the pectoralis major myocutaneous flap donor site.  

PubMed

The pectoralis major myocutaneous flap is considered a workhorse flap in the reconstruction of head and neck defects after cancer ablative surgeries and remains one of the most widely used reconstructive options. Complications at the donor site after the use of this flap, although rare, do occur and are usually restricted to minor infections, hematoma, and seroma formation. Metastasis to the flap donor site is a rare complication with limited documentation. Metastasis at the donor site usually follows local recurrence at the primary site, supporting the probable hypothesis of re-establishment of lymphatic drainage to the primary site by the flap pedicle. Tumor implantation, although a probable cause for metastasis at the donor site, cannot be confidently distinguished from other mechanisms, such as hematogenous spread or lymphogenous metastasis. This report describes a case that supports a seeding or tumor implantation mechanism of metastasis exclusively. PMID:24560174

Mohan, A Mathan; Balaguhan, B; Krishna, Vinod; Nagarjuna, Muralidhara

2014-07-01

108

Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites  

SciTech Connect

Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.

Bodaghi, Sohrab; Jia Rong [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States); Zheng Zhiming [HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States)], E-mail: zhengt@exchange.nih.gov

2009-03-30

109

Genetic interactions between the 5' and 3' splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing.  

PubMed Central

The YAG/ consensus sequence at the 3' end of introns (the slash indicates the location of the 3' splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5' end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5' /GUA with Y-3 of the 3' YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5' /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y-3 in the 3' YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y-3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5' /GUA and the GAG of U6 function in binding the 3' YAG/ during the second catalytic step.

Collins, C A; Guthrie, C

2001-01-01

110

Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.  

PubMed

Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis. PMID:23239986

Wappenschmidt, Barbara; Becker, Alexandra A; Hauke, Jan; Weber, Ute; Engert, Stefanie; Köhler, Juliane; Kast, Karin; Arnold, Norbert; Rhiem, Kerstin; Hahnen, Eric; Meindl, Alfons; Schmutzler, Rita K

2012-01-01

111

Analysis of 30 Putative BRCA1 Splicing Mutations in Hereditary Breast and Ovarian Cancer Families Identifies Exonic Splice Site Mutations That Escape In Silico Prediction  

PubMed Central

Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.

Hauke, Jan; Weber, Ute; Engert, Stefanie; Kohler, Juliane; Kast, Karin; Arnold, Norbert; Rhiem, Kerstin; Hahnen, Eric; Meindl, Alfons; Schmutzler, Rita K.

2012-01-01

112

Myopathy in a woman and her daughter associated with a novel splice site MTM1 mutation.  

PubMed

We have investigated a woman and her daughter with an early onset, slowly progressive myopathy. Muscle biopsy showed in both cases severe atrophy with marked fatty replacement. Frequent fibers with internalized nuclei were present but no typical features of centronuclear myopathy. There were also many fibers with deep invaginations of the plasma membrane. The presence of necklace fibers provided clue to correct genetic diagnosis. Both patients had a novel heterozygous splice site mutation in the myotubularin gene, MTM1 (c.867+1G>T). Analysis of MTM1 cDNA revealed that the mutation resulted in aberrant splicing with variable exon skipping. The expression of normal transcripts was markedly reduced and there was reduced expression of myotubularin protein. Although the expression of the allele without the mutation was reduced we did not obtain evidence of skewed X-chromosome inactivation. Other factors than skewed X-inactivation may cause allele inactivation and manifestation of severe myopathy in heterozygous carriers of pathogenic MTM1 mutations. PMID:22101172

Hedberg, Carola; Lindberg, Christopher; Máthé, Gyöngyvér; Moslemi, Ali-Reza; Oldfors, Anders

2012-03-01

113

A Crouzon syndrome synonymous mutation activates a 5{prime} splice site within the IIIC exon of the FGFR2 gene  

SciTech Connect

Crouzon syndrome, an autosomal dominant condition causing premature fusion of cranial structures, appears to be caused by mutations in the FGFR2 gene. Several mutations have been identified in the IIIc or bek exon that alter the amino acid sequence of the receptor in a zone known to be involved in ligand binding. In addition, a synonymous G to A transition has been described in three familial Crouzon syndrome cases (mutation at the third position of the alanine 344 codon). It has been suggested that this mutation may activate a cryptic 5{prime} or 3{prime} splice site. The significance of this latter mutation in Crouzon syndrome will be established only when it is known whether it does in fact affect splicing. If it does, prediction of the structure of the mutated receptor requires us to know whether a cryptic 5{prime} or a cryptic 3{prime} splice site has been activated. Ideally, splicing of the pre-mRNA would be studied in the cell type in which the mutated receptor is supposed to exert its effect. However, in our case this information is not available. An alternative strategy is to study splicing in cultured cells using cloned genes. The validity of this approach has been established in other disease systems, for example, thalassemias. 9 refs., 1 fig.

Gatto, F.D.; Breathnach, R. [INSERM, Nantes (France)

1995-06-10

114

Identification of a Novel RNA Splicing Pattern as a Basis of Restricted Cell Tropism of Erythrovirus B19  

Microsoft Academic Search

Prior studies on the transcription of erythrovirus B19 have identified a short leader sequence associated with all spliced viral transcripts. While some variability has been observed in the acceptor for this first intron, studies to date in both permissive and nonpermissive cell types have reported a unique splice donor site. In the semipermissive MB-02 cell line, we have found that

John Brunstein; Maria Söderlund-Venermo; Klaus Hedman

2000-01-01

115

Tissue expansion techniques to minimize morbidity of the anterolateral thigh perforator flap donor site.  

PubMed

Selection of any free flap donor site must not only meet the requirements of the recipient site but also minimize untoward sequela at the donor site itself. Although the anterolateral thigh (ALT) perforator flap is an ideal soft tissue donor site, a major drawback can be its nonesthetic appearance if a skin graft was needed. This detriment can be ameliorated by using traditional tissue expansion techniques. In a retrospective review over the past decade, 14 patients had ALT free flap donor site tissue expansion. These were subcategorized as pretransfer, concurrent, or posttransfer tissue expansion. In this group, mean ALT flap width was 12.2 ± 4.2 cm, which precluded direct donor site closure. Rectangular expanders were generally recommended. Multiple expanders are suggested for larger defects. The duration of expansion averaged 291.4 ± 163.9 days. The mean instilled volume ratio exceeded 2.43 ± 0.9 times the maximum vendor recommendation. Small skin graft residua were still left in four patients. Tissue expansion proved to be an important modality to consider for minimizing the stigmata of the skin grafted ALT free flap donor site. However, this process is time consuming and requires an additional surgical procedure. As such, this option must be reserved for the most motivated and compliant patients. PMID:23784789

Hallock, Geoffrey G

2013-11-01

116

Clinical comparative study of aquacel and paraffin gauze dressing for split-skin donor site treatment.  

PubMed

The management of split-thickness skin graft donor sites is targeted towards promoting the healing process, while minimizing adverse effects and complications. The aim of this study was to compare donor site treatment outcome between Aquacel, a carboxymethylcellulose-based hydrofiber dressing, and the standard mesh paraffin gauze dressing. The study included 23 adult patients. Half of the skin graft donor site in the proximal thigh was dressed with paraffin gauze and the rest with Aquacel. The results indicated that patients treated with Aquacel experienced significantly less pain and a more rapid rate of epithelialization compared with patients treated with mesh paraffin gauze dressing. Final scarring (ie, after the 1-year follow-up) was significantly better with the Aquacel dressing. We conclude that Aquacel dressing is superior to the standard mesh paraffin gauze dressing for split-thickness donor site area in pain relief, ease of treatment, promotion of epithelialization, and the quality of scarring. PMID:15269581

Barnea, Yoav; Amir, Aharon; Leshem, David; Zaretski, Arik; Weiss, Jerry; Shafir, Raphael; Gur, Eyal

2004-08-01

117

[Wound management with split skin flaps--donor sites. Covering with the moist gel Geliperm].  

PubMed

In 23 patients, donor sites of split-thickness skin grafts were treated with Geliperm Hydrogel, a swellable polyacrylamide agar. Healing duration, toleration, exudation and pain were all noted during the daily change of dressing. In 22 of the 23 cases, good healing was obtained after an average of 12.3 days. We feel that Geliperm is excellently suitable for covering the donor sites of split-thickness skin grafts. PMID:2328933

Sattler, G; Hagedorn, M

1990-02-20

118

Unusual patterns of exon skipping in Bruton tyrosine kinase are associated with mutations involving the intron 17 3' splice site.  

PubMed Central

Seven individuals with the diagnosis of X-linked agammaglobulinemia were analyzed for mutations in Bruton tyrosine kinase (Btk) gene at both the cDNA transcript and genomic DNA levels. In addition, maternal carrier status was determined in six of the seven families by examining X chromosome-inactivation patterns for B cells in comparison with other types of blood cells. Three categories of mutations were identified: (1) three patients have missense mutations in either the pleckstrin or SH2 domains of Btk; (2) three patients exhibit mutations at or near intron/exon splice sites, two of which represent inherited mutations within the kinase domain; and (3) one patient has inherited a 2.5-kb deletion with the loss of a DNA segment encoding three exons of the kinase domain. Variation in the lengths of Btk transcripts was evident in two patients with splice-site mutations and in the patient with the DNA deletion. Sequences of the different cDNA transcripts from the patients with 3' splice-site mutations reveal complex patterns of exon skipping involving from one to four exons of the kinase domain. These findings implicate 3' splice sites of the penultimate exon in the recognition or processing of upstream exons. Images Figure 2 Figure 3ab Figure 3c

Haire, R N; Ohta, Y; Strong, S J; Litman, R T; Liu, Y; Prchal, J T; Cooper, M D; Litman, G W

1997-01-01

119

Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site  

PubMed Central

All group II introns known to date fold into six functional domains. However, we recently identified an intron in Bacillus cereus ATCC 10987, B.c.I4, that splices 56?nt downstream of the expected 3? splice site in vivo (Tourasse et al. 2005, J. Bacteriol., 187, 5437–5451). In this study, we confirmed by ribonuclease protection assay that the 56-bp segment is part of the intron RNA molecule, and computational prediction suggests that it might form a stable stem-loop structure downstream of domain VI. The splicing of B.c.I4 was further investigated both in vivo and in vitro. Lariat formation proceeded primarily by branching at the ordinary bulged adenosine in domain VI without affecting the fidelity of splicing. In addition, the splicing efficiency of the wild-type intron was better than that of a mutant construct deleted of the 56-bp 3? extension. These results indicate that the intron has apparently adapted to the extra segment, possibly through conformational adjustments. The extraordinary group II intron B.c.I4 harboring an unprecedented extra 3? segment constitutes a dramatic example of the flexibility and adaptability of group II introns.

Stabell, Fredrik B.; Tourasse, Nicolas J.; Ravnum, Solveig; Kolst?, Anne-Brit

2007-01-01

120

Cultured allogeneic keratinocyte sheets accelerate healing compared to Op-site treatment of donor sites in burns.  

PubMed

Donor site treatment is a crucial issue in the treatment of extensive burns. In this single-blind, randomized study treatment of donor sites with a polyurethane dressing, Op-Site (Smith & Nephew, York, U.K.) is compared to treatment with allogeneic cultured keratinocyte sheets. Results show a mean healing time of 6.7 days with use of cultured keratinocyte sheets compared to mean healing time of 13.6 days with Op-Site treatment. Also, improvement in the comfort of patients as the result of less exudate formation and pain attenuation was noted. PMID:9404990

Duinslaeger, L A; Verbeken, G; Vanhalle, S; Vanderkelen, A

1997-01-01

121

Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end.  

PubMed Central

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5' splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5' splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5' end, suggesting that other factors than the U1 snRNA are involved in 5' splice site recognition. The importance of a consecutive complementarity between the 5' splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.

Lund, Mette; Kjems, J?rgen

2002-01-01

122

A Splice Site Mutation in Laminin-?2 Results in a Severe Muscular Dystrophy and Growth Abnormalities in Zebrafish  

PubMed Central

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-?2 gene (LAMA2). Laminin-?2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8–15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-?2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-?2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

Gupta, Vandana A.; Kawahara, Genri; Myers, Jennifer A.; Chen, Aye T.; Hall, Thomas E.; Manzini, M. Chiara; Currie, Peter D.; Zhou, Yi; Zon, Leonard I.; Kunkel, Louis M.; Beggs, Alan H.

2012-01-01

123

Probabilistic simple splicing systems  

NASA Astrophysics Data System (ADS)

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.

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

2014-06-01

124

Distant metastasis of intraosseous dentinogenic ghost cell tumour to the donor site of a bone graft  

PubMed Central

A dentinogenic ghost cell tumour (DGCT) is an extremely rare odontogenic tumour which is considered as a solid, neoplastic variant of calcifying odontogenic cyst. Intraosseous DGCTs are more aggressive than extraosseous DGCTs and have a high propensity for local recurrence. This report describes a case of a diagnosis of recurrent DGCT at the primary site and a distant donor site. A 25-year-old female patient visited a dental hospital for a complaint of facial swelling for the previous month. Incisional biopsy was performed and the specimen was diagnosed as DGCT. Partial mandibulectomy for tumour resection and iliac bone graft was performed. 2 years later, the tumour recurred on the mandible and iliac bone. The recurrent lesion on the donor site was diagnosed as metastasized DGCT. This report highlights the possibility of distant metastasis occurring at a graft donor site.

Park, H-R; Min, J-H; Huh, K-H; Yi, W-J; Heo, M-S; Lee, S-S; Cho, Y-A

2013-01-01

125

Graves’ Disease presenting as localized myxedema in a thigh donor graft site  

Microsoft Academic Search

Pretibial myxedema, exophthalmus, and thyroid acropachy are the classic manifestations of Graves’ Disease. However, myxedema in Graves’ Disease can occur in locations other than the pretibial surfaces. Furthermore, with systemic symptoms, localized myxedema may occur at sites of trauma or scarring. We describe a patient with localized myxedema on the thigh at the site of a donor skin graft as

Shani C. Missner; Evette W. Ramsay; Heather E. Houck; C. Lisa Kauffman

1998-01-01

126

Outcomes in head and neck reconstruction by surgical site and donor site  

PubMed Central

Objective Define surgical outcomes of specific donor sites for free tissue transfer in head and neck reconstruction. Design Retrospective cohort review Setting Academic tertiary care center. Patients A review of free tissue transfer procedures performed at a university-based tertiary care facility from October 2004 to April 2011. A total of 1051 patients underwent 6 types of free flaps: fasciocutaneous radial forearm (53%), osteocutaneous radial forearm (16%), rectus abdominus (11%), fibula (10%), anterior lateral thigh (7%), and latissimus dorsi (2%). Main Outcome Measures Demographic data was collected and outcomes measured were: length of hospital stay, flap viability, and major complications (infection, fistula, and hematoma). Results Of the 1051 flaps performed, the most common operative site was oral cavity (40% n=414) followed by hypopharynx/larynx (22%, n=234), cutaneous (20%, n=206), oropharynx (9%, n= 98), mid-face (7%, n= 76), and skull base (2%, n=23). The median hospital stay was 7.9 days (range 1-76) and the overall failure rate was 2.8%. Cutaneous defects required the shortest length of hospitalization (5.8 days, P< .0001), a low free flap failure rate (1.5%, n= 3), and limited major complications (6%, n= 12). Conversely, oropharynx defects were associated with the longest hospitalization (8.9 days). While midface defects had a high incidence of complications (15%, n= 11, P=.10). Defects above the angle of the mandible had higher overall complications when compared to below. Similarly, reconstruction for primary or recurrent cancer had a total failure rate of 2.5% while secondary reconstruction and radionecrosis had a failure rate of 4.0% (P=.29). Additionally, there was no statistical difference between outcomes based on donor site. Conclusions This review demonstrates that certain subsets of patients are at higher risk for complications after free tissue transfer. Patients undergoing free flap reconstruction for cutaneous defects have substantially shorter hospital stays and are at lower risk of flap complications, while reconstruction for radionecrosis or secondary reconstruction tend to have higher overall flap failure rates.

Frederick, JW; Sweeny, L; Carroll, WR; Peters, GE; Rosenthal, EL

2012-01-01

127

A novel splice site mutation in the EYA1 gene in a Korean family with branchio-oto (BO) syndrome.  

PubMed

Branchio-oto-renal (BOR) and branchio-oto (BO) syndromes are autosomal dominant hereditary disorders characterized by the presence of hearing loss and branchial fistulae and cysts, with (BOR syndrome) or without (BO syndrome) renal malformations of varying degrees of severity. Mutations in the human homologous of the Drosophila eyes absent (EYA1) gene are frequently the cause of BOR/BO syndrome. Here we describe a Korean family with BO syndrome; the proband had preauricular pit, cup-shaped auricles, branchial fistula, and hearing loss, without renal involvement. Molecular genetic study revealed a novel mutation occurring in the consensus acceptor splice site of intron 8 (c.868-2A > G) in the EYA1 gene. To the best of our knowledge, this is the first report of a splice site mutation in a family with BO syndrome without renal involvement, further extending the phenotypic-genotypic heterogeneity of BOR/BO syndrome. PMID:18763178

Kwon, Min-Jung; Boo, Sung Hyun; Kim, Hee-Jin; Cho, Yang-Sun; Chung, Won-Ho; Hong, Sung Hwa

2009-06-01

128

Reconstruction of complex defects of the parotid region using a lateral thoracic wall donor site.  

PubMed

Radical treatment of parotid neoplasms may lead to complex parotid defects that present functional and aesthetic reconstructive challenges. We report our experience using the lateral thoracic wall as a single donor site. Between 2003 and 2009, four patients with malignant tumours in the parotid gland underwent radical parotidectomy and simultaneous reconstruction using a perforator latissimus dorsi cutaneous free flap (de-epithelialized and entire skin paddle in two cases each). A thoracodorsal nerve graft was used in all cases to replace the intraglandular branches of the facial nerve. Costal grafts were used for mandibular reconstruction in two patients. All patients underwent postoperative physiotherapy. No donor-site complication occurred and all treatments achieved good aesthetic results. All patients recovered nearly complete symmetry at rest and partial facial mimetic function. The lateral thoracic wall is a good donor site for the reconstruction of complex parotid defects. PMID:23245945

Biglioli, Federico; Pedrazzoli, Marco; Rabbiosi, Dimitri; Colletti, Giacomo; Colombo, Valeria; Frigerio, Alice; Autelitano, Luca

2013-04-01

129

Donor-site morbidity of the inferior gluteal musculocutaneous flap for breast reconstruction in teenagers.  

PubMed

The purpose of this study is to objectively evaluate donor-site morbidity of the inferior gluteal musculocutaneous flap in teenagers. All cases of breast reconstruction performed between 1996 and 2005 using an inferior gluteal flap were reviewed. Flap size, weight, and pedicle origin were noted. Donor-site morbidity was assessed for scarring, contour deformity, muscle function, and sensation. The charts of 15 patients were reviewed, and 6 patients were available for further investigation. The average flap size and weight were 17 cm x 7 cm and 430 g, respectively. All patients had a well-concealed scar with minimal buttock asymmetry. Sensory assessment showed some degree of hypoesthesia in the territory of the posterior femoral cutaneous nerve in all patients. There was no functional loss. Donor-site morbidity of the inferior gluteal musculocutaneous flap is largely related to posterior thigh hypoesthesia despite preservation of the posterior femoral cutaneous nerve. PMID:18046140

Dupéré, Sophie; Bergeron, Léonard; Bortoluzzi, Patricia; Del-Duca, Tina; Caouette-Laberge, Louise

2007-12-01

130

ASSESSMENT OF DONOR SITE MORBIDITY FOR FREE RADIAL FOREARM OSTEOCUTANEOUS FLAPS  

PubMed Central

Purpose Assessment of donor site morbidity and recipient site complications following free radial forearm osteocutaneous flap (FRFOCF) harvest and evaluation of patient perceived upper limb disability for free radial forearm osteocutaneous versus fasciocutaneous flaps (FRFF). Methods First a case series was undertaken of 218 patients who underwent an FRFOCF at two tertiary referral centers between February 1998 and November 2010. Outcomes included forearm donor site morbidity and recipient site complications. Second, the disability of the arm, shoulder, and hand (DASH) questionnaire assessing patient perceived arm disability was administered by phone to 60 consecutive patients who underwent an FRFOCF or FRFF. Results Mean patient age was 63 years with male predominance (62.8%). Median bone length harvested was 8 cm (range, 3–12 cm) with prophylactic plating of the radius following harvest. Donor site morbidity included fracture (1 patient, 0.5%) and sensory neuropathy (5 patients, 2.3%). Mean DASH scores were comparative between groups and to established normative values. Mandibular malunion rate was 3.2% and hardware extrusion at the recipient site occurred in 15.6%. Conclusion Reluctance to perform FRFOCF by surgeons usually centers on concerns regarding potential donor site morbidity and adequacy of available bone stock; however, we identified minimal objective or patient perceived donor site morbidity or recipient site complications following harvest of FRFOCFs. Mild wrist weakness and stiffness are common but do not impede ability to perform activities of daily living. Data from this and other reports suggest this flap is particularly useful for midfacial and short segment mandibular reconstruction.

Sinclair, Catherine F.; Gleysteen, John P.; Zimmermann, Terence M.; Wax, Mark K.; Givi, Babak; Schneider, Daniel; Rosenthal, Eben L.

2014-01-01

131

Heteroduplex formation and S1 digestion for mapping alternative splicing sites  

Microsoft Academic Search

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pair- ing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks

E. N. Ferreira; M. C. R. Rangel; P. B. Pineda; D. O. Vidal; A. A. Camargo; S. J. Souza; D. M. Carraro

2008-01-01

132

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)  

PubMed Central

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

2013-01-01

133

The missing puzzle piece: splicing mutations  

PubMed Central

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.

Lewandowska, Marzena A

2013-01-01

134

Management of split skin graft donor sites-results of a national survey.  

PubMed

The authors wished to obtain a 'snapshot' of the range of practice in the management of split skin graft donor sites in the British Isles. Material/Methods Questionnaires were sent to all British consultants and locum consultant plastic surgeons on July 1, 2006. Of the 357 questionnaires, 279 were returned (a response rate of 78%). Results Alginates were the most popular dressings, especially in adult donor sites - first choice for 167 respondents (60%). Adhesive fabrics were less popular - first choice for small adult donor areas for 46 respondents (16%). Plastic film dressings and Biobrane were even less popular - being the first choice for small and large donor areas, respectively, in children (for approximately 5% of respondents). Ten percent of respondents said they avoid paraffin gauze and another 10% avoid plastic film dressings in all cases. Five percent avoid hydrocolloid and another 5% avoid adhesive fabric in all cases. Conclusion on the basis of these results, the authors feel that any future study of donor-site dressings should incorporate the most commonly used dressing (alginate) as a control. PMID:22099851

Geary, P M; Tiernan, E

2012-01-01

135

The use of a collagen sponge\\/living cell composite material to treat donor sites in burn patients  

Microsoft Academic Search

The objective of this study was to examine the safety and efficacy of bilayered cellular matrix, (OrCel™) Ortec International, Inc., New York, NY in facilitating timely wound closure of split-thickness donor sites in severely burned patients. We utilized a matched pairs design; each patient had two designated donor sites of equivalent surface area and depth. Sites were randomized to receive

Joseph Still; Paul Glat; Paul Silverstein; John Griswold; David Mozingo

2003-01-01

136

The intronic splicing code: multiple factors involved in ATM pseudoexon definition  

PubMed Central

Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5? splice site-like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP-binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP-mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans-acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.

Dhir, Ashish; Buratti, Emanuele; van Santen, Maria A; Luhrmann, Reinhard; Baralle, Francisco E

2010-01-01

137

Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection  

PubMed Central

TDP-43 is a critical RNA-binding factor associated with pre-mRNA splicing in mammals. Its expression is tightly autoregulated, with loss of this regulation implicated in human neuropathology. We demonstrate that TDP-43 overexpression in humans and mice activates a 3? untranslated region (UTR) intron, resulting in excision of the proximal polyA site (PAS) pA1. This activates a cryptic PAS that prevents TDP-43 expression through a nuclear retention mechanism. Superimposed on this process, overexpression of TDP-43 blocks recognition of pA1 by competing with CstF-64 for PAS binding. Overall, we uncover complex interplay between transcription, splicing, and 3? end processing to effect autoregulation of TDP-43.

Avendano-Vazquez, S. Erendira; Dhir, Ashish; Bembich, Sara; Buratti, Emanuele; Proudfoot, Nicholas; Baralle, Francisco E.

2012-01-01

138

Exon 3 of the alpha folate receptor gene contains a 5' splice site which confers enhanced ovarian carcinoma specific expression.  

PubMed

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT-PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1-chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5' and 3' deleted variants of the P1-CAT construct were analyzed by RT-PCR of the exogenous transcripts and reporter activity. A 5' splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma. PMID:11478943

Galmozzi, E; Tomassetti, A; Sforzini, S; Mangiarotti, F; Mazzi, M; Nachmanoff, K; Elwood, P C; Canevari, S

2001-07-27

139

Reduction of Donor Site Morbidity of Free Radial Forearm Flaps: What Level of Evidence Is Available?  

PubMed Central

Background: The radial forearm free flap (RFFF) is the most commonly used free flap in head and neck reconstructive surgery. However, despite excellent results with respect to the site of reconstruction, donor site morbidity cannot be neglected. This review summarizes the current state of knowledge and analyzes the level of evidence with regard to perioperative management of the reduction of RFFF donor site morbidity. Methods: The medical Internet source PubMed was screened for relevant articles. All relevant articles were tabulated according to the levels of scientific evidence, and the available methods for reduction of donor site morbidity are discussed. Results: Classification into levels of evidence reveals 3 publications (1.5%) with level I (randomized controlled trials), 29 (14.0%) with level II (experimental studies with no randomization, cohort studies, or outcome research), 3 (1.5%) with level III (systematic review of case-control studies or individual case-control studies), 121 (58.7%) with level IV (nonexperimental studies, such as cross-sectional trials, case series, case reports), and 15 (7.3%) with level V (narrative review or expert opinion without explicit critical appraisal). Thirty-five (17.0%) articles could not be classified, because they focused on a topic other than donor site morbidity of the RFFF. Conclusions: Although great interest has been expressed with regard to reducing the donor site morbidity of the workhorse flap in microvascular reconstruction procedures, most publications fail to provide the hard facts and solid evidence characteristic of high-quality research.

Loeffelbein, Denys J.; Al-Benna, Sammy; Steinstrasser, Lars; Satanovskij, Robin M.; Rohleder, Nils H.; Mucke, Thomas; Wolff, Klaus-Dietrich; Kesting, Marco R.

2012-01-01

140

Graves' disease presenting as localized myxedema in a thigh donor graft site.  

PubMed

Pretibial myxedema, exophthalmus, and thyroid acropachy are the classic manifestations of Graves' Disease. However, myxedema in Graves' Disease can occur in locations other than the pretibial surfaces. Furthermore, with systemic symptoms, localized myxedema may occur at sites of trauma or scarring. We describe a patient with localized myxedema on the thigh at the site of a donor skin graft as the initial presentation of Graves' Disease. PMID:9810913

Missner, S C; Ramsay, E W; Houck, H E; Kauffman, C L

1998-11-01

141

Steric antisense inhibition of AMPA receptor Q/R editing reveals tight coupling to intronic editing sites and splicing  

PubMed Central

Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site—the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site.

Penn, Andrew C.; Balik, Ales; Greger, Ingo H.

2013-01-01

142

Tumour Transfer to Bone Graft Donor Site: A Case Report and Review of the Literature of the Mechanism of Seeding  

PubMed Central

Purpose. Transmission of malignant tumour cells to a bone graft donor site is a rare complication of bone grafting.We report a case of seeding of malignant fibrous histiocytoma from the femur to a pelvic bone graft donor site. Discussion. We review the literature, discuss the possible mechanism of tumour transfer and offer advice aimed at avoiding this complication.

Dias, Richard G.; Carter, Simon R.; Grimer, Robert J.; Tillman, Roger M.

2000-01-01

143

Tra2-Mediated Recognition of HIV-1 5? Splice Site D3 as a Key Factor in the Processing of vpr mRNA  

PubMed Central

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3? splice site (3?ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5?ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESEvpr) localized between exonic splicing silencer ESSV and 5?ss D3. The ESEvpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESEvpr. Further analyses revealed that ESEvpr supports the binding of U1 snRNA at 5?ss D3, allowing bridging interactions across the upstream exon with 3?ss A2. In line with this, an increase or decrease in the complementarity of 5?ss D3 to the 5? end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3?ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5?ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3?ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.

Erkelenz, Steffen; Poschmann, Gereon; Theiss, Stephan; Stefanski, Anja; Hillebrand, Frank; Otte, Marianne; Stuhler, Kai

2013-01-01

144

A Dual Reporter Splicing Assay Using HaloTag-containing Proteins.  

PubMed

To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag-luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species. Preliminary experiments confirmed that the dual reporter minigene assay can provide estimates of overall splicing efficiency based on the levels of protein products. We then used the minigene assay to analyze a case of chronic granulomatous disease that was caused by a G>C mutation at position +5 in the 5'-splice donor site of intron 5 of the CYBB gene. We found that the G>C mutation affected CYBB mRNA splicing by changing a delicate balance of splicing efficiencies of introns 4, 5, and 6. PMID:23136623

Oshima, Koichi; Nagase, Takahiro; Imai, Kohsuke; Nonoyama, Shigeaki; Obara, Megumi; Mizukami, Tomoyuki; Nunoi, Hiroyuki; Kanegane, Hirokazu; Kuribayashi, Futoshi; Amemiya, Shin; Ohara, Osamu

2012-01-01

145

Aberrant 3? splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization  

PubMed Central

The frequency distribution of mutation-induced aberrant 3? splice sites (3?ss) in exons and introns is more complex than for 5? splice sites, largely owing to sequence constraints upstream of intron/exon boundaries. As a result, prediction of their localization remains a challenging task. Here, nucleotide sequences of previously reported 218 aberrant 3?ss activated by disease-causing mutations in 131 human genes were compared with their authentic counterparts using currently available splice site prediction tools. Each tested algorithm distinguished authentic 3?ss from cryptic sites more effectively than from de novo sites. The best discrimination between aberrant and authentic 3?ss was achieved by the maximum entropy model. Almost one half of aberrant 3?ss was activated by AG-creating mutations and ?95% of the newly created AGs were selected in vivo. The overall nucleotide structure upstream of aberrant 3?ss was characterized by higher purine content than for authentic sites, particularly in position ?3, that may be compensated by more stringent requirements for positive and negative nucleotide signatures centred around position ?11. A newly developed online database of aberrant 3?ss will facilitate identification of splicing mutations in a gene or phenotype of interest and future optimization of splice site prediction tools.

Vorechovsky, Igor

2006-01-01

146

In vitro analysis of splice site mutations in the CLCN1 gene using the minigene assay.  

PubMed

Mutations in the chloride channel gene CLCN1 cause the allelic disorders Thomsen (dominant) and Becker (recessive) myotonia congenita (MC). The encoded protein, ClC-1, is the primary channel that mediates chloride (Cl-) conductance in skeletal muscle. Mutations in CLCN1 lower the channel's threshold voltage, leading to spontaneous action potentials that are not coupled to neuromuscular transmission and resulting in myotonia. Over 120 mutations in CLCN1 have been described, 10% of which are splicing defects. Biological specimens suitable for RNA extraction are not always available, but obtaining genomic DNA for analysis is easy and non-invasive. This is the first study to evaluate the pathogenic potential of novel splicing mutations using the minigene approach, which is based on genomic DNA analysis. Splicing mutations accounted for 23% of all pathogenic variants in our cohort of MC patients. Four were heterozygous mutations in four unrelated individuals, belonging to this cohort: c.563G>T in exon 5; c.1169-5T>G in intron 10; c.1251+1G>A in intron 11, and c.1931-2A>G in intron 16. These variants were expressed in HEK 293 cells, and aberrant splicing was verified by in vitro transcription and sequencing of the cDNA. Our findings confirm the need to further investigate the nature of rearrangements associated with this class of mutations and their effects on mature transcripts. In particular, splicing mutations predicted to generate in-frame transcripts may generate out-of-frame mRNA transcripts that do not produce functional ClC-1. Clinically, incomplete molecular evaluation could lead to delayed or faulty diagnosis. PMID:24452722

Ulzi, Gianna; Sansone, Valeria A; Magri, Francesca; Corti, Stefania; Bresolin, Nereo; Comi, Giacomo P; Lucchiari, Sabrina

2014-05-01

147

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

PubMed Central

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

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

2012-01-01

148

Which dressing do donor site wounds need?: study protocol for a randomized controlled trial  

PubMed Central

Background Donor site wounds after split-skin grafting are rather 'standard' wounds. At present, lots of dressings and topical agents for donor site wounds are commercially available. This causes large variation in the local care of these wounds, while the optimum 'standard' dressing for local wound care is unclear. This protocol describes a trial in which we investigate the effectiveness of various treatment options for these donor site wounds. Methods A 14-center, six-armed randomized clinical trial is being carried out in the Netherlands. An a-priori power analysis and an anticipated dropout rate of 15% indicates that 50 patients per group are necessary, totaling 300 patients, to be able to detect a 25% quicker mean time to complete wound healing. Randomization has been computerized to ensure allocation concealment. Adult patients who need a split-skin grafting operation for any reason, leaving a donor site wound of at least 10 cm2 are included and receive one of the following dressings: hydrocolloid, alginate, film, hydrofiber, silicone dressing, or paraffin gauze. No combinations of products from other intervention groups in this trial are allowed. Optimum application and changes of these dressings are pursued according to the protocol as supplied by the dressing manufacturers. Primary outcomes are days to complete wound healing and pain (using a Visual Analogue Scale). Secondary outcomes are adverse effects, scarring, patient satisfaction, and costs. Outcome assessors unaware of the treatment allocation will assess whether or not an outcome has occurred. Results will be analyzed according to the intention to treat principle. The first patient was randomized October 1, 2009. Discussion This study will provide comprehensive data on the effectiveness of different treatment options for donor site wounds. The dressing(s) that will prevail in effectiveness, satisfaction and costs will be promoted among clinicians dealing with such patients. Thus, we aim to contribute a well-designed trial, relevant to all clinicians involved in the care for donor site wounds, which will help enhance uniformity and quality of care for these patients. Trial registration http://www.trialregister.nl, NTR1849. Date registered: June 9, 2009

2011-01-01

149

The properties of the "ideal" donor site dressing: results of a worldwide online survey  

PubMed Central

Summary Split skin grafting is a widely used technique for reconstructing skin defects. Although a vast number of different coverage options for donor sites have become available in daily clinical practice, no optimum dressing material has been found to date. For this reason, we conducted a globally-distributed online survey to poll for the properties of such an “ideal” donor site dressing, possibly leading to an improved clinically-driven direction of future wound dressing developments. A total of 69 respondents from 34 countries took part in the questionnaire, resulting in a response rate of 13.8% (69/500) over a 1-month period. The majority of respondents rated the characteristics of an “ideal” donor site dressing to be either “essential” or “desirable” as follows: lack of adhesion to the wound bed (“essential”: 31/69, 44.9%; “desirable”: 30/69, 43.5%); pain-free dressing changes (“essential”: 38/69, 55.1%; “desirable”: 30/69, 43.5%); absorbency (“essential”: 27/69, 39.1%; “desirable”: 33/69, 47.8%); ease of removal (“essential”: 37/69, 53.6%; “desirable”: 27/69, 39.13%). With regard to the desired frequency of dressing changes, respondents preferred “no dressing change until the donor site has healed” (51/69, 73.9%) in the majority of cases, followed by “twice weekly” (10/69, 14.5%), “alternate days” (5/69, 7.2%) and “daily” (3/69, 4.3%). With regard to the design of the dressing material, the majority of participants preferred a one-piece (composite) dressing product (44/69, 63.8%). The majority of respondents also denied the current availability of an “ideal” donor site dressing (49/69, 71%). The strength of this study was the remarkable geographic distribution of responses; all parts of the world were included and participated. We believe that this globally conducted online survey has polled for the properties of the “ideal” donor site dressing and possibly will lead to an improved clinically-driven direction of future wound dressing development.

Lars, P. Kamolz L.P.; Giretzlehner, M.; Trop, M.; Parvizi, D.; Spendel, S.; Schintler, M.; Justich, I.; Wiedner, M.; Laback, C.; Lumenta, D.B..

2013-01-01

150

Comparison of scalp and abdomen as split-thickness skin graft donor sites for aural stenosis repair.  

PubMed

To evaluate and compare the scalp and the abdomen as split-thickness skin graft donor sites for aural stenosis repair. A total of 34 patients with aural stenosis were included in the study. All the patients underwent meatoplasty using split-thickness skin grafts. Among them, the skin graft donor site was the scalp in 11 patients and the abdomen in the other 23 patients. The surgical team followed the patients in the outpatient department for at least 6 months after surgery. Evaluations concerned healing of the donor site, hair regeneration of the donor site, survival of split-thickness skin grafts, reoccurrence of aural stenosis and hair growth in the ear canal. The incidences of reoccurrence of aural stenosis in the two groups were compared. Subjective scar evaluation of the donor sites was performed using the Patient Scar Assessment Scale (PASA). The scale items were pain, itching, color, stiffness, thickness and irregularity. All the scalp and abdominal donor sites healed well with no sign of infection. Hair regrowth and reepithelialization was observed at all the scalp donor sites. Pink discoloration was observed at the scalp donor sites in six patients 2-3 months after surgery and disappeared 6-9 months after surgery. Scars were observed at the scalp donor sites in two patients 6 months after surgery. No alopecia was observed at the scalp donor sites. The scars and pink discoloration were hidden in the hair. Scars and/or discoloration were observed at all the abdominal donor sites 12 months after surgery. All the scalp and abdominal skin grafts survived with no sign of infection. Hair growth was observed in the ear canals in two patients in the scalp group. The incidences of reoccurrence of aural stenosis were 0 % (0/23) in the abdominal group and 9.1 % (1/11) in the scalp group, respectively (Chi square test, p > 0.05). The PASA values about color, stiffness, thickness and irregularity were higher in the abdominal group than in the scalp group (Mann-Whitney U test, p < 0.001). The PASA values about pain and itching were the same (Mann-Whitney U test, p > 0.05). The scalp meets most requirements of an ideal donor site of skin grafts for aural stenosis. The advantages of scalp as a donor site include easy accessibility in the operative field, simple postoperative care, low risk of infection, rapid wound healing, minimal interference with rehabilitation, and minimal scar formation. PMID:24057102

Du, Qiang; Zhang, Tianyu

2014-08-01

151

Resurfacing glabrous skin defects in the hand: the thenar base donor site.  

PubMed

Defects of the glabrous skin surfaces of the palm and fingers result from numerous causes including larger fingertip injuries, unhealed burns, and after surgery for diverse pathologies. The qualities of glabrous skin are specifically tailored to the functional requirements of high-shear strength and robustness. Despite these unique properties, graft reconstruction of defects in the glabrous regions of the hand is frequently achieved with skin from nonglabrous donor sites such as the medial forearm. Nonglabrous skin has a poor color and texture match for such applications and is frequently associated with tender and unsightly donor scars. We describe our experiences of harvesting full-thickness grafts from the glabrous skin centered over the proximal flexion crease at the level of the metacarpophalangeal joint of the thumb. We have utilized this site to harvest skin grafts of up to 2 cm in width for the resurfacing of small-sized to medium-sized defects on the palmar surfaces of the hands and fingers in 28 patients under both traumatic and elective circumstances. The skin has an excellent type-match to the defect and is quick and easy to harvest due to its adjacent location to the defect. The donor scar matures quickly, and as it lies along the thumb base crease, it runs along one of the least used contact surfaces, thereby limiting the potential discomfort associated with FTSG harvest sites from other areas. Patient satisfaction with the procedure has been high, and it represents a useful alternative to traditional nonglabrous skin graft donor sites for small-sized to medium-sized defects. PMID:24637743

Milner, Chris S; Thirkannad, Sunil M

2014-06-01

152

Evaluation of an oxygen-diffusion dressing for accelerated healing of donor-site wounds.  

PubMed

Accelerating the healing process and reducing pain during healing are beneficial for the following reasons: faster return to work, lower risk of wound infection, improved quality of life, and possibly reduced need for analgesia. This clinical study assessed the effectiveness of a new oxygen-diffusion dressing (OxyBand; Oxyband Technologies, St. Louis, MO) compared with standard Xeroform gauze dressings (Convidien, Mansfield, MA), in the care of skin-graft donor sites in burn patients. Time to healing was the primary endpoint, and pain scores and cosmetic outcome were also assessed. This was a prospective, randomized, controlled study of burn patients undergoing harvesting of two donor sites. Patients were followed at predetermined time points for 30 to 45 days to determine the time to reepithelialization, cosmetic appearance, and pain. Subjects were adult burn patients with less than 30% TBSA burns admitted to the burn center, who required excision and grafting. Twenty patients were enrolled, of whom 17 completed the study. Average age was 35 years. Average burn size was 9.2% TBSA. Patients underwent harvesting of split-thickness skin grafts with one donor wound dressed with OxyBand and the other dressed in Xeroform gauze. Wounds were inspected and photographed on postoperative days 4 and 8, and then every 2 days until the donor wounds were healed. Pain scores at each site were also collected at these visits (rated by patients on a scale from 0 to 10). Mean time to wound healing for OxyBand was 9.3 ± 1.7 days; for Xeroform, 12.4 ± 2.7 days (P < .001). Pain scores were lower (P < .01) at the OxyBand site compared with the Xeroform site at all time points during postoperative days 4 to 12. There was no difference in the cosmetic outcome of the wounds at 30 to 45 days postoperatively. This study revealed a decrease in the time to healing and in pain at donor sites dressed with an oxygen-diffusion dressing. PMID:23877142

Lairet, Kimberly F; Baer, David; Leas, Michelle L; Renz, Evan M; Cancio, Leopoldo C

2014-01-01

153

Solid phase electron donors control denitrification in groundwater at agricultural sites  

NASA Astrophysics Data System (ADS)

Increased concentrations of nitrate in groundwater caused by agricultural use of chemical and organic fertilizers are a concern because of possible risks to environmental and human health. At many sites, these problems are mitigated by natural attenuation of nitrate as a result of microbially mediated denitrification of nitrate to nitrogen gas. Recent studies have clarified the factors affecting the rates and extents of denitrification in groundwater in agricultural areas. Intensive studies were conducted by the US Geological Survey to study agricultural chemicals in California, Nebraska, Washington, and Maryland using laboratory analyses, field measurements, and flow and transport modeling for monitoring well transects (0.5 to 2.5 km in length) and vertical profiles (0 to 50 m in depth). Groundwater analyses included major ion chemistry, dissolved gases, nitrogen and oxygen stable isotopes, and atmospheric age-tracers. Sediments were analyzed for concentrations of potential electron donors for denitrification, including reduced iron and sulfur, and organic carbon. Geochemical data and mass balance calculations indicated that solid-phase electron donors were an important factor controlling denitrification at these sites. To examine the generality of this result, a mathematical model of vertical flux of water, oxygen, and nitrate was developed and applied at these study sites along with 2 new study sites in Iowa and Mississippi and 8 additional sites from previous studies in Nebraska, Texas, Minnesota, Wisconsin, North Carolina, Maryland (2 sites), and New York. Model results confirmed the importance of solid phase electron donors. The normalized reaction rates on an electron flux basis tended to increase with depth from the shallow oxygen reduction zone to the underlying nitrate reduction zone. The pattern of higher rates at depth is consistent with a reaction rate controlled by solid phase donors that are depleted under oxidizing conditions near the surface and in greater supply at depth. The eventual depth and rate of migration of nitrate and oxygen in aquifers will depend on the concentrations and reactivities of solid electron donor phases currently in the reduced zones.

Green, C. T.; Liao, L.; Bekins, B. A.; Bohlke, J. K.

2011-12-01

154

Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron.  

PubMed Central

We previously reported that exon skipping in vivo due to point mutations in the 5' splice site (5'ss) signal of an internal mammalian exon can be prevented by coexpression of U1 small nuclear RNAs, termed shift-U1s, with complementarity to sequence upstream or downstream of the mutated site. We now show by S1 nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not necessarily of the down stream intron. This indicates that the normal 5'ss sequence acts as an enhancer for splicing of the upstream intron, that it owes this activity to base pairing with U1, and that the enhancer activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the upstream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and the 5'ss sequence is not necessary for accurate splicing of the downstream intron. These findings are discussed in relation to the coordinate selection exon termini proposed by the exon definition model.

Hwang, D Y; Cohen, J B

1996-01-01

155

Uroporphyrinogen decarboxylase: a splice site mutation causes the deletion of exon 6 in multiple families with porphyria cutanea tarda.  

PubMed Central

Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree. Images

Garey, J R; Harrison, L M; Franklin, K F; Metcalf, K M; Radisky, E S; Kushner, J P

1990-01-01

156

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

PubMed Central

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.

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

2010-01-01

157

Redirecting splicing with bifunctional oligonucleotides  

PubMed Central

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5? splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.

Brosseau, Jean-Philippe; Lucier, Jean-Francois; Lamarche, Andree-Anne; Shkreta, Lulzim; Gendron, Daniel; Lapointe, Elvy; Thibault, Philippe; Paquet, Eric; Perreault, Jean-Pierre; Abou Elela, Sherif; Chabot, Benoit

2014-01-01

158

The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA.  

PubMed

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize. PMID:23739895

Regulski, Michael; Lu, Zhenyuan; Kendall, Jude; Donoghue, Mark T A; Reinders, Jon; Llaca, Victor; Deschamps, Stephane; Smith, Andrew; Levy, Dan; McCombie, W Richard; Tingey, Scott; Rafalski, Antoni; Hicks, James; Ware, Doreen; Martienssen, Robert A

2013-10-01

159

The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA  

PubMed Central

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.

Regulski, Michael; Lu, Zhenyuan; Kendall, Jude; Donoghue, Mark T.A.; Reinders, Jon; Llaca, Victor; Deschamps, Stephane; Smith, Andrew; Levy, Dan; McCombie, W. Richard; Tingey, Scott; Rafalski, Antoni; Hicks, James; Ware, Doreen; Martienssen, Robert A.

2013-01-01

160

The 100-kda U5 snRNP protein (hPrp28p) contacts the 5' splice site through its ATPase site.  

PubMed Central

To identify splicing factors in proximity of the 5' splice site (5'SS), we followed a crosslinking profile of site-specifically modified, photoreactive RNA substrates. Upon U4/U5/U6 snRNP addition, the 5'SS RNA crosslinks in an ATP-dependent manner to U6 snRNA, an unidentified protein p27, and the 100-kDa U5 snRNP protein, a human ortholog of an ATPase/RNA helicase yPrp28p. The 5'SS:hPrp28p crosslink maps to the highly conserved TAT motif in proximity of the ATP-binding site in hPrp28p. We propose that hPrp28p acts as a helicase to unwind the 5'SS:U1 snRNA duplex, and at the same time as a 5'SS translocase, which, upon NTP-dependent conformational change, positions the 5'SS for pairing with U6 snRNA within the spliceosome. This repositioning of the 5'SS takes place regardless of whether the 5'SS is originally duplexed with U1 snRNA.

Ismaili, N; Sha, M; Gustafson, E H; Konarska, M M

2001-01-01

161

Trans-splicing and alternative-tandem-cis-splicing: two ways by which mammalian cells generate a truncated SV40 T-antigen.  

PubMed Central

The early SV40 BstXI-BamHI (Bst/Bam) DNA fragment encodes exclusively for the second exon of the large T-antigen and contains the intact small t-antigen intron. Rat cells transformed by the p14T, a construct that carries the Bst/Bam DNA fragment as a tail-to-head tandem duplication, synthesize a truncated T-antigen (T1-antigen) without having a direct equivalent at the DNA level. Formation of the T1-mRNA occurs by means of two distinct mechanisms: alternative-tandem-cis-splicing and trans-splicing. To generate the T1-mRNA the cells utilize a cryptic 5' splice site, located within the second exon of the large T-antigen and the regular small t-antigen 3' splice site. Since these splice sites are in an inverted order two Bst/Bam transcripts are required to generate one T1-mRNA molecule. For alternative-tandem-cis-splicing the cells utilize a 4.4 kb pre-mRNA that contains the sequence of the entire Bst/Bam tandem repeat. The proximal Bst/Bam segment provides the 5' donor splice site and the distal segment the 3' acceptor site. This requires that the pre-mRNA not be cleaved after the RNA polymerase II has passed the polyadenylation signal of the proximal Bst/Bam DNA segment. Synthesis of the 4.4 kb pre-mRNA was demonstrable by RT-PCR but not by Northern blot analysis. For trans-splicing, the cells utilize two separate pre-mRNA molecules. One transcript provides the cryptic 5' splice donor site and the other the 3' splice acceptor site. To demonstrate this a three base pair deletion was introduced into the proximal Bst/Bam segment of the p14T DNA (p14Tdelta-3) as a marker, destroying the recognition site for Pf/MI restriction enzyme. This deletion allowed the differentiation between the proximal and distal Bst/Bam segment. RT-PCR analysis and DNA sequencing confirmed that the p14Tdelta-3 transformed cells generate the T1-mRNA by intra- and inter-molecular RNA splicing.

Eul, J; Graessmann, M; Graessmann, A

1996-01-01

162

Efficacy of Quilting Sutures and Fibrin Sealant Together for Prevention of Seroma in Extended Latissimus Dorsi Flap Donor Sites  

PubMed Central

Background The extended latissimus dorsi flap is important for breast reconstruction. Unfortunately, donor site seroma is the most common complication of extended latissimus dorsi flap for breast reconstruction. Although using fibrin sealant in the donor site reduces the rate of seroma formation, donor site seroma remains a troublesome complication. The purpose of this study was to analyze the effectiveness of the combination of quilting sutures and fibrin sealant in the latissimus dorsi donor site for the prevention of seroma. Methods Forty-six patients who underwent breast reconstruction with extended latissimus flap were enrolled in the study. The patients received either fibrin sealant (group 1, n=25) or a combination of fibrin sealant and quilting sutures (group 2, n=21) in the extended latissimus dorsi donor site. Outcome measures were obtained from the incidence, volume of postoperative seroma, total drainage amount, indwelling period of drainage, and duration of hospital stay. Results The incidence of seroma was 76% in group 1 and 42.9% in group 2 (P=0.022). We also found significant reductions in seroma volume (P=0.043), total drainage amount (P=0.002), indwelling period of drainage (P=0.01), and frequency of aspiration (P=0.043). The quilting sutures did not affect the rate of drainage, tube reinsertion, or hospital stay. Conclusions The use of quilting sutures combined with fibrin sealant on the latissimus dorsi flap donor site is helpful for reducing the overall seroma volume, frequency of aspiration, and total drainage amount.

Shin, In Soo; Lee, Dong Won

2012-01-01

163

Treatment of burns and donor sites with human allogeneic keratinocytes grown on acellular pig dermis.  

PubMed

The absence of a dermal component predisposes cultured epidermal sheets to instability, contractibility, and makes them difficult to handle. In order to overcome these drawbacks, we developed recombined human/pig skin (RHPS) composed of human keratinocytes cultured on cell-free pig dermis. The original intention to prepare a permanent skin substitute composed of xenodermis and autologous epidermis was not achieved, but it has been proved that RHPS can serve as an effective, ready to use keratinocyte delivery system when applied 'upside-down', i.e. with epidermal cells facing the wound surface. The keratinocyte layer establishes a direct contact with the wound bed, while the dermal layer mechanically protects the wound. Twenty deep dermal burns were grafted with RHPS: 13 (65%) healed completely in 4-14 days, three (15%) healed partially and four (20%) did not heal. Of five full thickness burn wounds only one healed after repeated RHPS grafting within 18 days. Thirty-one (100%) donor sites treated with any of the three forms of RHPS, subconfluent, confluent meshed or confluent unmeshed, healed within 6-8 days compared with 14-18 days in control sites. Seven donor sites (100%) of immunodeficient patients with prolonged wound healing epithelialized in 7-10 days under RHPS compared with 32-90 days in areas treated with tulle gras and dry gauze. PMID:9217823

Matousková, E; Bucek, S; Vogtová, D; Veselý, P; Chaloupková, A; Broz, L; Singernová, H; Pavlíková; Königová, R

1997-06-01

164

Umbilical hernia--a potential donor-site complication of fat injection laryngoplasty.  

PubMed

Injection laryngoplasty with autologous fat appears to be an effective and simple technique for the treatment of patients with glottic insufficiency in comparison with other surgical techniques. Despite of its advantages, associated complications have also been reported, including immediate donor-site morbidity (eg, hematoma and abscess), fat extrusion of the injection site, and delayed manifestation of vocal granuloma or overinjected vocal folds. In this article, a patient suffering from accidental injury to the deep abdominal fascia without peritoneal penetration in the fat harvest procedure is presented. Three months after the fat injection laryngoplasty, an umbilical hernia was proved to occur via the clinical imaging. Several etiologies are supposed to induce the herniation of intraabdominal structures, including surgeon's incaution, abdominal obesity, intense wound inflammation and fibrosis, and the native weak point of the abdominal wall around the umbilicus. This case provides information that overdepth and negligence in fat harvest may injure the deep abdominal fascia, then possibly causing the umbilical hernia as a delayed donor-site complication. PMID:23177750

Chiu, Feng-Shiang; Lin, Yaoh-Shiang; Chang, Ying-Nan; Lee, Jih-Chin

2012-11-01

165

The free vascularised iliac crest tissue transfer: donor site complications associated with eighty-two cases.  

PubMed

Seventy-eight patients who had undergone a total of 82 free vascularised iliac crest tissue transfers were reviewed to determine the incidence of donor site complications. The most frequent problems encountered were early postoperative pain and long term sensory changes. Major complications such as femoral neuropathy and incisional hernia formation were encountered infrequently. More serious potential complications are discussed. In general, the functional loss associated with the free vascularised iliac crest tissue transfer was found to be acceptable, but the inclusion of a skin paddle was noted to be associated with a greater incidence of sensory changes, hernia formation and contour abnormalities. PMID:1562853

Forrest, C; Boyd, B; Manktelow, R; Zuker, R; Bowen, V

1992-01-01

166

Use of the hydrocolloidal dressing duoderm for skin donor sites for burns.  

PubMed

We have made a study of the use of Duoderm hydroactive sterile occlusive dressing on 10 patients for skin donor sites. Its therapeutic efficacy is evident and the dressing enhances the wound debridement and accelerates the re-epithelialization, with complete healing in 8.5 days on the average. In comparison with a conventional dressing with paraffin gauze, Duoderm allows a more rapid re-epithelialization. In addition, the new skin is softer, smoother and more homogeneous. Duoderm is also easy to use and is well tolerated by the patients. PMID:3074956

Donati, L; Vigano, M

1988-01-01

167

Human palatal and tuberosity mucosa as donor sites for ridge augmentation.  

PubMed

Since different clinical outcomes of periodontal bilaminar surgeries using the palate or the maxillary tuberosity as connective tissue (CT) donor sites have been observed, tissues grafted with CT from the palate or the tuberosity 1 year after surgical procedures for ridge augmentation were compared with nongrafted tissues by using morphologic and molecular methods. Collagen content and matrix metalloproteinases 1 and 2 expression were similar in tissues and cultured fibroblasts from the palate and tuberosity, although with interindividual differences. In contrast, differences in collagen cross-linking and maturation in the tuberosity fibroblasts were observed, suggesting a possible role in determining hyperplastic responses in some patients. PMID:24600654

Dellavia, Claudia; Ricci, Giano; Pettinari, Letizia; Allievi, Cristina; Grizzi, Fabio; Gagliano, Nicoletta

2014-01-01

168

An alternatively spliced region of the human hexabrachion contains a repeat of potential N-glycosylation sites.  

PubMed Central

We have cloned and sequenced two cDNA molecules that code for parts of two forms of human hexabrachion. The smaller clone has a sequence that corresponds to the previously published sequence of a cDNA clone coding for a part of chicken hexabrachion [Jones, F. S., Burgoon, M. P., Hoffman, S., Crossin, K. L., Cunningham, B. A. & Edelman, G. M. (1988) Proc. Natl. Acad. Sci. USA 85, 2186-2190]. It has eight consecutive domains similar to the type III homology units from fibronectin, several epidermal growth factor repeats, and a domain similar to the beta and gamma chains of fibrinogen. The larger clone has 5' and 3' ends that are identical to the smaller clone but also has an alternatively spliced 1.9-kilobase internal segment. The unique segment contains remarkable repeats of potential glycosylation sites and an additional seven type III homology units. Images

Gulcher, J R; Nies, D E; Marton, L S; Stefansson, K

1989-01-01

169

A Novel Splice Site Mutation in the SERPING1 Gene Leads to Haploinsufficiency by Complete Degradation of the Mutant Allele mRNA in a Case of Familial Hereditary Angioedema.  

PubMed

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal-dominant and life-threatening disorder caused by mutations in SERPING1 gene. It is characterized by attacks of angioedema involving the skin and/or the mucosa of the upper airways, as well as the intestinal mucosa. Here we report the case of a patient with HAE-C1INH without family history of angioedema. By sequencing the SERPING1 gene we detected a novel mutation (c.1249?+?5G?>?A) affecting the 5' donor splice site in intron 7. We analyzed the SERPING1 cDNA expecting a defect in splicing process but only the wild type allele was detected. SNP analysis of the cDNA sequence demonstrated that only one of the two alleles was present, indicating that the mRNA from the mutated allele was completely degraded. This study reinforces the concept of incomplete penetrance of this disorder since the patients' mother never presented any sign of angioedema despite carrying the same mutation. PMID:24760113

Colobran, Roger; Pujol-Borrell, Ricardo; Hernández-González, Manuel; Guilarte, Mar

2014-07-01

170

A splice variant of Dp71 lacking the syntrophin binding site is expressed in early stages of human neural development  

Microsoft Academic Search

Dp71, a 71 kDa C-terminal isoform of dystrophin, is the major product of the DMD gene in brain. Two alternatively spliced transcripts of Dp71 were amplified by RT-PCR from different areas of human fetal neural tissue. Both transcripts were spliced out of exons 71 and 78. The shorter transcript was also alternatively spliced of exons 72–74, a region comprising the

Marina Ceccarini; Giovanni Rizzo; Giuseppina Rosa; Cristiana Chelucci; Pompeo Macioce; Tamara C Petrucci

1997-01-01

171

Full thickness skin grafts from the groin: donor site morbidity and graft survival rate from 50 cases  

PubMed Central

Objectives Full thickness skin grafts (FTSG) offer several advantages; they are esthetically superb, have less postoperative shrinkage, and offer minimal postoperative pain and scar formation at the donor site. As a donor site of FTSG, the groin offers a relatively large area of skin with high elasticity. The aim of this study was to evaluate FTSG from the groin for reconstruction in oral and maxillofacial surgery. Materials and Methods In a retrospective study, 50 patients (27 males, 23 females) who received FTSG from the groin were evaluated for their operation records, clinical photography, and medical records. Results The width of skin from the groin was distributed from 2-8 cm (mean: 5.1 cm) at the donor site, while the long axis length was distributed from 3-13 cm (mean: 7.4 cm). A high number of patients, 47 patients (94%) out of 50, showed good healing at the donor site. Wound impairment was seen in 3 patients (6%), minor wound dehiscence in 2 patients, and severe wound dehiscence in 1 patient. In the recipient site, delayed healing was observed in 2 patients (4%). Conclusion FTSG from the groin to repair soft tissue defects in reconstruction surgery is a good method due to the relatively big size of the graft, decreasing morbidity at the donor site, and higher graft survival rates.

Kim, Somi; Chung, Seung-Won

2013-01-01

172

A Spontaneous Fatp4/Scl27a4 Splice Site Mutation in a New Murine Model for Congenital Ichthyosis  

PubMed Central

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3?-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.

Tao, Jianning; Koster, Maranke I.; Harrison, Wilbur; Moran, Jennifer L.; Beier, David R.; Roop, Dennis R.; Overbeek, Paul A.

2012-01-01

173

Mitogen-activated protein kinase phosphorylation of splicing factor 45 (SPF45) regulates SPF45 alternative splicing site utilization, proliferation, and cell adhesion.  

PubMed

The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38? also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H(2)O(2), UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation. PMID:22615491

Al-Ayoubi, Adnan M; Zheng, Hui; Liu, Yuying; Bai, Tao; Eblen, Scott T

2012-07-01

174

Role of helical constraints of the EBS1-IBS1 duplex of a group II intron on demarcation of the 5' splice site.  

PubMed

Recognition of the 5' splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem-loop of domain 1 and a complementary sequence at the 3' end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5? intron, we probed the solution structure of the ID3 stem-loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5? and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5' splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1-IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5? and O.i. introns and may help to fine-tune elements of recognition in group II introns. PMID:24243113

Popovic, Milena; Greenbaum, Nancy L

2014-01-01

175

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing - a prospective randomized clinical trial on skin graft donor sites: platelet concentrate and keratinocytes on donor sites  

PubMed Central

Background Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC?+?K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. Results Healing time was reduced from 13.9?±?0.5 days (mean?±?SEM) in the control group to 7.2?±?0.2 days in the PC group (P?

2013-01-01

176

The use of a collagen sponge/living cell composite material to treat donor sites in burn patients.  

PubMed

The objective of this study was to examine the safety and efficacy of bilayered cellular matrix, (OrCel) Ortec International, Inc., New York, NY in facilitating timely wound closure of split-thickness donor sites in severely burned patients. We utilized a matched pairs design; each patient had two designated donor sites of equivalent surface area and depth. Sites were randomized to receive a single treatment of either OrCel or the standard dressing Biobrane-L (Bertek Pharmaceuticals) Sugarland, TX. The results demonstrate that OrCel was more effective in facilitating timely wound closure of split-thickness skin donor sites than Biobrane-L. The healing time for OrCel sites was significantly shorter than for sites treated with Biobrane-L. This acceleration of wound healing was clinically important in enabling earlier recropping. OrCel sites also exhibited reduced scarring. Therefore, treatment of donor site wounds with OrCel is well tolerated, promotes more rapid healing, and results in reduced scarring when compared with conventional therapy with Biobrane-L. PMID:14636761

Still, Joseph; Glat, Paul; Silverstein, Paul; Griswold, John; Mozingo, David

2003-12-01

177

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing*  

PubMed Central

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5? splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

Guan, Wuxiang; Huang, Qinfeng; Cheng, Fang; Qiu, Jianming

2011-01-01

178

Homologous SV40 RNA trans-splicing: a new mechanism for diversification of viral sequences and phenotypes.  

PubMed

Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5' donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5'ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5'ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes. PMID:24178438

Eul, Joachim; Patzel, Volker

2013-11-01

179

Use of lyophilized bovine collagen for split-thickness skin graft donor site management.  

PubMed

Donor site management after split-thickness skin graft applications can have problems such as late healing and pain. Many dressing methods and medical applications are reported to solve these problems but none of them were ideal. In this study we aimed to promote epithelisation and remove pain earlier with using lyophilized bovine collagen (gelfix spray). According to our results, epithelisation time for the gelfix group was earlier than control group (9.09 days mean and 11.2 days mean for control group (p<0.05)). Pain relief was determined by visual analogue pain scale. In the gelfix group, there was pain relief up to 40 h from the operation. There were no differences between groups for scarring 30 and 90 days after surgery. PMID:18407418

Uygur, Fatih; Evinc, Rahmi; Ulkur, Ersin; Celikoz, Bahattin

2008-11-01

180

Activated endothelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1  

PubMed Central

Vascular cell adhesion molecule-1 (VCAM-1) is induced on endothelial cells by inflammatory cytokines, and binds mononuclear leukocytes through the integrin very late antigen-4 (VLA-4) (alpha 4 beta 1). This adhesion pathway has been implicated in a diverse group of physiological and pathological processes, including B cell development, leukocyte activation and recruitment to sites of inflammation, atherosclerosis, and tumor cell metastasis. The major form of VCAM-1 (VCAM-7D) has seven extracellular immunoglobulin (Ig)-like domains, of which the three NH2-terminal domains (domains 1-3) are similar in amino acid sequence to domains 4-6. By functional analysis of VCAM-7D relative to VCAM-6D (a minor 6-domain form of VCAM-1 in which domain 4 is deleted because of alternative splicing), and chimeric constructs between VCAM-1 and its structural relative intercellular adhesion molecule-1 (ICAM-1), we show that either the first or the homologous fourth domain of VCAM-1 is required for VLA-4-dependent adhesion. Either of these binding sites can function in the absence of the other. When both are present, cell binding activity is increased relative to monovalent forms of the molecule. The homologous binding regions appear to have originated by internal duplication of a portion of a monovalent ancestral gene, before the mammalian radiation. Thus VCAM-1 exemplifies evolution of a functionally bivalent cell-cell adhesion molecule by intergenic duplication. We have also produced a new class of anti-VCAM- 1 monoclonal antibodies that block domain 4-dependent adhesion, and demonstrate that both binding sites participate in the adhesion function of VCAM-1 on endothelial cells in vitro. Therefore both sites must be blocked in clinical, animal, or in vitro studies depending on the use of anti-VCAM-1 antibodies to inactivate the VCAM-1/VLA-4 adhesion pathway.

1992-01-01

181

Donor-site morbidity of the inferior gluteal artery perforator flap for breast reconstruction in teenagers  

PubMed Central

BACKGROUND/OBJECTIVE: Few options, apart from the buttock area, are available for autologous breast reconstruction in thin teenagers. The aim of the present study was to objectively evaluate and compare donor-site morbidity of the inferior gluteal artery perforator (IGAP) flap with that of the previously described inferior gluteal musculocutaneous flap. METHOD: A retrospective review of all IGAP flaps for breast reconstruction performed in teenagers between June 2006 and April 2011 at the Centre Hospitalier Universitaire Sainte-Justine, Montreal, Quebec, was performed. Patients were invited to undergo a specific physical evaluation and complete a questionnaire on aesthetic and functional outcomes. RESULTS: Thirteen records and 11 photographic charts were reviewed. Lateral buttock flattening was noticeable in nine of 11 cases. Three patients experienced some degree of inferior displacement of the gluteal crease. All six patients available for the appointment presented with a zone of dysesthesia or hypoesthesia in the territory of the operated buttock and/or posterior thigh. No motor impairment was found. The questionnaire, completed by eight patients, revealed that six were satisfied or very satisfied with the surgery. Appearance of the operated buttock was rated 3.4 on a scale from 1 to 5 (5 = normal) compared with the normal side. CONCLUSIONS: The IGAP flap remains a suitable option for breast reconstruction in slim teenagers. Similar to the myocutaneous flap, the major donor-site morbidity of the IGAP flap remains sensory impairment involving the posterior femoral cutaneous nerve. There is, however, less visible lateral depression when it is harvested as a perforator flap.

Godbout, Emilie; Farmer, Lucie; Bortoluzzi, Patricia; Caouette Laberge, Louise

2013-01-01

182

NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML.  

PubMed

Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics. PMID:24574459

Adamia, Sophia; Bar-Natan, Michal; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bach, Christian; Pevzner, Samuel; Calimeri, Teresa; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Galinsky, Ilene; Mathews, Steven; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Tenen, Daniel G; Stone, Richard M; Griffin, James D

2014-05-01

183

Utilization of a Continuous External Tissue Expansion System to Assist in Primary Closure of a Large Anterolateral Thigh Donor Site Defect  

PubMed Central

Primary closure of a large anterolateral thigh (ALT) flap donor site defect with the assistance of an external tissue expansion system is presented. The dimensions of this donor site (12?cm × 40?cm) and its percentage of leg circumference (34%) would make this site likely to require skin grafting or further flap coverage based on the results of previously published literature.

Silver, Andrew G.; Baynosa, Richard C.

2014-01-01

184

Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity  

Microsoft Academic Search

Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5-FU), thereby limiting the efficacy of the therapy. It has been suggested that patients suffering from 5-FU toxicides due to a low activity of DPD are genotypically heterozygous for a mutant allele of the gene encoding DPD. In this study we investigated the cDNA and

A. B. P. Van Kuilenburg; P. Vreken; L. V. A. M. Beex; R. Meinsma; H. Van Lenthe; R. A. De Abreu; A. H. Van Gennip

1997-01-01

185

Lethal autosomal recessive epidermolytic ichthyosis due to a novel donor splice-site mutation in KRT10.  

PubMed

Epidermolytic ichthyosis (EI; MIM 113800), previously named bullous congenital ichthyosiform erythroderma or epidermolytic hyperkeratosis, is a rare and clinically variable defect of cornification characterized by generalized erythema, erosions, scaling and easily breaking blisters that become less frequent later in life while hyperkeratosis increases. EI is caused by dominant mutations in either KRT1 or KRT10, encoding keratin 1 (K1) and keratin 10 (K10), respectively. Usually, mutations are missense substitutions into the highly conserved ?-helical rod domains of the proteins. However, three inbred pedigrees in which EI is transmitted as a recessive trait due to KRT10 null mutations have been described. PMID:20302579

Covaciu, C; Castori, M; De Luca, N; Ghirri, P; Nannipieri, A; Ragone, G; Zambruno, G; Castiglia, D

2010-06-01

186

Randomized placebo-controlled human pilot study of cold atmospheric argon plasma on skin graft donor sites.  

PubMed

Cold atmospheric plasma has already been shown to decrease the bacterial load in chronic wounds. However, until now it is not yet known if plasma treatment can also improve wound healing. We aimed to assess the impact of cold atmospheric argon plasma on the process of donor site healing. Forty patients with skin graft donor sites on the upper leg were enrolled in our study. The wound sites were divided into two equally sized areas that were randomly assigned to receive either plasma treatment or placebo (argon gas) for 2 minutes. Donor site healing was evaluated independently by two blinded dermatologists, who compared the wound areas with regard to reepithelialization, blood crusts, fibrin layers, and wound surroundings. From the second treatment day onwards, donor site wound areas treated with plasma (n = 34) showed significantly improved healing compared with placebo-treated areas (day 1, p = 0.25; day 2, p = 0.011; day 3, p < 0.001; day 4, p < 0.001; day 5, p = 0.004; day 6, p = 0.008; day 7, p = 0.031). Positive effects were observed in terms of improved reepithelialization and fewer fibrin layers and blood crusts, whereas wound surroundings were always normal, independent of the type of treatment. Wound infection did not occur in any of the patients, and no relevant side effects were observed. Both types of treatment were well tolerated. The mechanisms contributing to these clinically observed effects should be further investigated. PMID:23937657

Heinlin, Julia; Zimmermann, Julia L; Zeman, Florian; Bunk, Wolfram; Isbary, Georg; Landthaler, Michael; Maisch, Tim; Monetti, Roberto; Morfill, Gregor; Shimizu, Tetsuji; Steinbauer, Julia; Stolz, Wilhelm; Karrer, Sigrid

2013-01-01

187

RNA Splicing in a New Rhabdovirus from Culex Mosquitoes?†  

PubMed Central

Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

Kuwata, Ryusei; Isawa, Haruhiko; Hoshino, Keita; Tsuda, Yoshio; Yanase, Tohru; Sasaki, Toshinori; Kobayashi, Mutsuo; Sawabe, Kyoko

2011-01-01

188

A randomized, prospective, parallel group study of laparoscopic versus laparoendoscopic single site donor nephrectomy for kidney donation.  

PubMed

Few prospective, randomized studies have assessed the benefits of laparoendoscopic single site donor nephrectomy (LESS-DN) over laparoscopic donor nephrectomy (LDN). Our center initiated such a trial in January 2011, following subjects randomized to LESS-DN versus LDN from surgery through 5 years postdonation. Subjects complete recovery/satisfaction questionnaires at 2, 6 and 12 months postdonation; transplant recipient outcomes are also recorded. One hundred subjects (49 LESS-DN, 51 LDN) underwent surgery; donor demographics were similar between groups, and included a predominance of female, living-unrelated donors, mean age of 47 years who underwent left donor nephrectomy. Operative parameters (overall time, time to extraction, warm ischemia time, blood loss) were similar between groups. Conversion to hand-assist laparoscopy was required in 3 LESS-DN (6.1%) versus 2 LDN (3.9%; p?=?0.67). Questionnaires revealed that 97.2% of LESS-DN versus 79.5% of LDN (p?=?0.03) were 100% recovered by 2 months after donation. No significant difference was seen in satisfaction scores between the groups. Recipient outcomes were similar between groups. Our randomized trial comparing LESS donor nephrectomy to LDN confirms that LESS-DN offers a safe alternative to conventional LDN in terms of intra- and post-operative complications. LDN and LESS-DN offer similar recovery and satisfaction after donation. PMID:24934732

Aull, M J; Afaneh, C; Charlton, M; Serur, D; Douglas, M; Christos, P J; Kapur, S; Del Pizzo, J J

2014-07-01

189

A novel family with recessive von Willebrand disease due to compound heterozygosity for a splice site mutation and a missense mutation in the von Willebrand factor gene  

Microsoft Academic Search

We report a new family with autosomal recessive von Willebrand disease (VWD) in which the propositus was compound heterozygous for a missense mutation in exon 42 (G7085T, C2362F) and a C?A splice site mutation in intron 13 of the von Willebrand factor (VWF) gene. The propositus had factor VIII:C ?20 IU\\/dl, VWF antigen 5–7 IU\\/dl and ristocetin cofactor activity 15

Giancarlo Castaman; Elisabetta Novella; Evelina Castiglia; Jeroen C. J Eikenboom; Francesco Rodeghiero

2002-01-01

190

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection  

NASA Astrophysics Data System (ADS)

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

191

Evolution of splicing regulatory networks in Drosophila.  

PubMed

The proteome expanding effects of alternative pre-mRNA splicing have had a profound impact on eukaryotic evolution. The events that create this diversity can be placed into four major classes: exon skipping, intron retention, alternative 5' splice sites, and alternative 3' splice sites. Although the regulatory mechanisms and evolutionary pressures among alternative splicing classes clearly differ, how these differences affect the evolution of splicing regulation remains poorly characterized. We used RNA-seq to investigate splicing differences in D. simulans, D. sechellia, and three strains of D. melanogaster. Regulation of exon skipping and tandem alternative 3' splice sites (NAGNAGs) were more divergent than other splicing classes. Splicing regulation was most divergent in frame-preserving events and events in noncoding regions. We further determined the contributions of cis- and trans-acting changes in splicing regulatory networks by comparing allele-specific splicing in F1 interspecific hybrids, because differences in allele-specific splicing reflect changes in cis-regulatory element activity. We find that species-specific differences in intron retention and alternative splice site usage are primarily attributable to changes in cis-regulatory elements (median ?80% cis), whereas species-specific exon skipping differences are driven by both cis- and trans-regulatory divergence (median ?50% cis). These results help define the mechanisms and constraints that influence splicing regulatory evolution and show that networks regulating the four major classes of alternative splicing diverge through different genetic mechanisms. We propose a model in which differences in regulatory network architecture among classes of alternative splicing affect the evolution of splicing regulation. PMID:24515119

McManus, C Joel; Coolon, Joseph D; Eipper-Mains, Jodi; Wittkopp, Patricia J; Graveley, Brenton R

2014-05-01

192

The U11-48K Protein Contacts the 5? Splice Site of U12-Type Introns and the U11-59K Protein? †  

PubMed Central

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5? splice sites (5?ss). This interaction did not require branch point recognition and was sensitive to 5?ss mutations, suggesting that 48K interacts with the 5?ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5?ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5?ss, as well as functionally important interactions within the U11/U12 di-snRNP.

Turunen, Janne J.; Will, Cindy L.; Grote, Michael; Luhrmann, Reinhard; Frilander, Mikko J.

2008-01-01

193

Long-term functional donor site morbidity of the free radial forearm flap in head and neck cancer survivors  

PubMed Central

Background To assess the functional donor site morbidity of the forearm free flap in patients surviving at least 2 years after ablative head and neck cancer surgery in a tertiary care centre. Methods This study involved nine long-term survivors (2 year post-operative) who had forearm free flaps to reconstruct head and neck defects. All flaps were raised from the non-dominant arm. The non-donor side acted as a control for all patients. Objective measurements were as follows: grip, tip pinch and key pinch strength measured with dynamometers; flexion, extension, radial and ulnar deviation and pronation and supination range of motion at the wrist measured with goniometry; A timed manual dexterity task was performed with a grooved pegboard test, and sensation of the radial nerve was tested with Semmes Weinstein monofilaments. Subjective measurements included a validated patient questionnaire of hand function and opinions of scar appearance as well as a validated scar assessment from two different observers. Results Pronation at the wrist, manual dexterity and sensation were found to be significantly reduced in the donor side compared to the non-donor side. Inter-rater agreement between the two observers was found to be poor, except for an acceptable correlation between overall scar opinions. No correlations were found between any subjective or objective items or between the patient’s and the observers’ subjective evaluations. Conclusions Donor site morbidity can be demonstrated with objective testing however this is accepted and well tolerated by head and neck cancer patients.

2014-01-01

194

The scalp as a donor site for split-thickness skin graft: a rare complication case report.  

PubMed

The scalp is a useful and reliable donor site for the paediatric burn population that can be harvested several times with minimal morbidity. However, the scalp cannot be used as skin graft donor site with impunity. Scalp alopecia and chronic folliculitis can be observed among the complications. In these cases, the reconstruction phase offers different surgical procedures such as primary closure, staged excision or tissue expansion. We report the case of a patient (29-years-old), treated 20 years ago for second-degree burns covering up to 20% total body surface area (TBSA) by using thin split-thickness skin grafts of his scalp. As a teenager, he developed multiple episodes of folliculitis at the donor site of the scalp and then of recurrent abscesses, resistant to all existing medical treatments. Surgical treatment consisted in the skin excision of his scalp donor site which was immediately covered by a thin split-thickness skin graft. Four months after surgery, the patient was satisfied with the functional and aesthetic result. PMID:21300581

Robert, N; May, P; Binder, J P; Revol, M; Servant, J M

2011-05-01

195

Comparison of the ionic silver-containing hydrofiber and paraffin gauze dressing on split-thickness skin graft donor sites.  

PubMed

The split-thickness skin graft (STSG) donor site dressing has been an inconclusive topic. Each of the Hydrofiber (Aquacel, ConvaTec A Bristol-Myers Squibb Company, Deeside, UK) and silver dressings have applied in many types of wound care with favorable outcomes. Our study compared the ionic silver-containing Hydrofiber dressing and paraffin gauze dressing. The subjects were randomized into group A: ionic silver-containing Hydrofiber and group B: paraffin gauze. From February 2006 to 2007, 20 donor sites were recorded. The mean donor site surface area was 145.5 cm2 (group A) and 135.8 cm2 (group B). The completed re-epithelization day was 7.90 and 11.20 days, respectively (P = 0.031). The average pain score at rest were 0.74 and 0.80, respectively (P = 0.894). The average pain score on dressing removal were 3.12 and 4.70, respectively (P = 0.027). There was no infection or seroma in both groups. In conclusion, ionic silver-containing Hydrofiber dressing can reduce STSG donor site pain and promote re-epithelization compared to paraffin gauze dressing. PMID:19325350

Lohsiriwat, Visnu; Chuangsuwanich, Apirag

2009-04-01

196

MutPred Splice: machine learning-based prediction of exonic variants that disrupt splicing  

PubMed Central

We have developed a novel machine-learning approach, MutPred Splice, for the identification of coding region substitutions that disrupt pre-mRNA splicing. Applying MutPred Splice to human disease-causing exonic mutations suggests that 16% of mutations causing inherited disease and 10 to 14% of somatic mutations in cancer may disrupt pre-mRNA splicing. For inherited disease, the main mechanism responsible for the splicing defect is splice site loss, whereas for cancer the predominant mechanism of splicing disruption is predicted to be exon skipping via loss of exonic splicing enhancers or gain of exonic splicing silencer elements. MutPred Splice is available at http://mutdb.org/mutpredsplice.

2014-01-01

197

Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding.  

PubMed Central

The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate contents of 24M(-1.)min(-1), 0.8M(-1.)min(-1) and 0.54M(-1.)min(-1) respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0M(-1.)min(-1). The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement or arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (deltaG')for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete activation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35mM compared with 10mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichromism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.

Adak, S; Mazumder, A; Banerjee, R K

1996-01-01

198

A novel splice site mutation of the thiazide-sensitive NaCl cotransporter gene in a Japanese patient with Gitelman syndrome.  

PubMed

Gitelman syndrome (GS, MIM 263800) is an inherited disorder characterized by metabolic alkalosis with hypokalemia, hypomagnesemia, and hypocalciuria. The genetic abnormalities causing GS are known to lie in the thiazide-sensitive NaCl cotransporter (TSC), which is expressed in the distal tubule of the kidney. The TSC gene, located at chromosome 16, consists of 26 exons and encodes the protein containing 12 putative transmembrane domains with long intracellular amino and carboxy termini. Most of the abnormalities identified in GS were missense mutations, distributed throughout the TSC gene without a hot spot. A 42-year-old Japanese man was introduced for close examination of hypokalemia. In renal clearance studies using furosemide or thiazide, chloride clearance was increased after furosemide but not after thiazide administration. Furthermore, the distal fractional chloride reabsorption was dramatically decreased by furosemide but not thiazide administration, suggesting a defect in the distal tubule. We then analyzed the TSC gene to confirm the diagnosis of GS, and identified a novel G to T mutation at the acceptor splice site preceding exon 14, resulting in disruption of a conventional 3'AG consensus splice site. Abnormal splicing by this mutation is predicted to cause the formation of truncated TSC with a partial deletion of the transmembrane domain, which will loose the function of transporter. In conclusion, we have identified a unique novel splice site mutation of the TSC gene in GS. The predicted structure of this mutant TSC can conceivably cause an impairment of the transporter activity and thereby be responsible for the development of GS in our patient. PMID:15481849

Iida, K; Hanafusa, M; Maekawa, I; Kudo, T; Takahashi, K; Yoshioka, S; Kishimoto, M; Iguchi, G; Tsukamoto, T; Okimura, Y; Kaji, H; Chihara, K

2004-09-01

199

Selection of Alternative 5? Splice Sites: Role of U1 snRNP and Models for the Antagonistic Effects of SF2/ASF and hnRNP A1  

PubMed Central

The first component known to recognize and discriminate among potential 5? splice sites (5?SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5?SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5?SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5?SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5?SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5?SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5?SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

Eperon, Ian C.; Makarova, Olga V.; Mayeda, Akila; Munroe, Stephen H.; Caceres, Javier F.; Hayward, Daniel G.; Krainer, Adrian R.

2000-01-01

200

Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging  

PubMed Central

Background The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Results Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Conclusions Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.

2014-01-01

201

Whole exome sequencing identifies a novel splice-site mutation in ADAMTS17 in an Indian family with Weill-Marchesani syndrome  

PubMed Central

Purpose Weill-Marchesani syndrome (WMS) is a rare connective tissue disorder, characterized by short stature, microspherophakic lens, and stubby hands and feet (brachydactyly). WMS is caused by mutations in the FBN1, ADAMTS10, and LTBP2 genes. Mutations in the LTBP2 and ADAMTS17 genes cause a WMS-like syndrome, in which the affected individuals show major features of WMS but do not display brachydactyly and joint stiffness. The main purpose of our study was to determine the genetic cause of WMS in an Indian family. Methods Whole exome sequencing (WES) was used to identify the genetic cause of WMS in the family. The cosegregation of the mutation was determined with Sanger sequencing. Reverse transcription (RT)–PCR analysis was used to assess the effect of a splice-site mutation on splicing of the ADAMTS17 transcript. Results The WES analysis identified a homozygous novel splice-site mutation c.873+1G>T in a known WMS-like syndrome gene, ADAMTS17, in the family. RT–PCR analysis in the patient showed that exon 5 was skipped, which resulted in the deletion of 28 amino acids in the ADAMTS17 protein. Conclusions The mutation in the WMS-like syndrome gene ADAMTS17 also causes WMS in an Indian family. The present study will be helpful in genetic diagnosis of this family and increases the number of mutations of this gene to six.

Shah, Mohd Hussain; Bhat, Vishwanath; Shetty, Jyoti S.

2014-01-01

202

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

PubMed Central

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.

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

2013-01-01

203

Conserved RNA secondary structures promote alternative splicing  

Microsoft Academic Search

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alter- native splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexi- bility, splice-site selection in higher eukaryotes

PETER J. SHEPARD; KLEMENS J. HERTEL

2008-01-01

204

Does distance matter? Variations in alternative 3? splicing regulation  

PubMed Central

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.

Akerman, Martin; Mandel-Gutfreund, Yael

2007-01-01

205

TFPI? is an Active Alternatively Spliced Form of TFPI Present in Mice but not in Humans  

PubMed Central

Background Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor procoagulant activity produced as two alternatively spliced isoforms, TFPI? and TFPI?, which differ in domain structure and mechanism for cell surface association. 3’ RACE was used to search for new TFPI isoforms. TFPI?, a new alternatively spliced form of TFPI was identified and characterized. Methods The tissue expression, cell surface association and anticoagulant activity of TFPI? were characterized and compared to TFPI? and TFPI? through studies of mouse and human tissues and expression of recombinant proteins in CHO cells. Results TFPI? is produced by alternative splicing using the same 5’ splice donor site as TFPI? and a 3’ splice acceptor site 187 nucleotides beyond the stop codon of TFPI? in exon 8. The resulting protein has the first two Kunitz domains connected to an 18 amino acid C-terminal region specific to TFPI?. TFPI? mRNA is differentially produced in mouse tissues but is not encoded within the human TFPI gene. When expressed in CHO cells, TFPI? is secreted into conditioned media and effectively inhibits tissue factor procoagulant activity. Conclusions TFPI? is a third alternatively spliced form of TFPI widely expressed in mouse tissues but not made by human tissues. It contains the first two Kunitz domains and is a secreted, rather than a cell surface associated protein. It is a functional anticoagulant and may partially explain the resistance of mice to coagulopathy in tissue factor mediated models of disease.

Maroney, Susan A.; Ferrel, Josephine P.; Collins, Maureen L.; Mast, Alan E.

2013-01-01

206

Topical radiant heating in wound healing: an experimental study in a donor site wound model*.  

PubMed

The importance of temperature in the wound-healing process is rapidly being recognised as a novel way in which to manipulate the wound-healing environment. In this study, we aimed to investigate the direct effect of topical radiant heating (TRH), using a novel bandaging system (Warm-Up, Arizant Health care Inc., Eden Prairie MN, USA; Augustine Medical, USA), on wound healing at a physiological and cellular level. Experimental bandages were positioned over split-thickness skin graft donor site wounds of 12 patients undergoing graft harvesting from the anterior thigh. The experimental group (n=6) underwent intermittent heating for 5 hours (three 1-hour heating cycles at 38 degrees C, separated by two 1-hour rest periods), whilst the control group (n=6) received no radiant heating. Physiological blood-flow recordings both in the control group and the topical radiant heat cohort were undertaken using Laser Doppler Imaging (LDI). Skin biopsies were obtained at identical time points, and immunohistochemical analysis was undertaken using antibodies against neutrophils (NP57), lymphocytes (CD3) and macrophages (CD68). We found that TRH significantly increased local dermal blood flow (P<0.001) by up to 100% in both injured and intact skin. Furthermore, this increase in flow was associated with a significant (P<0.05) increase in CD3 immunoreactivity on day 1 postoperatively. This study demonstrates that TRH increases local blood flow and lymphocyte (CD3) extravasation, and we postulate that these changes may enhance local innate immunity within the healing wound environment. PMID:16722872

Khan, Aadil A; Banwell, Paul E; Bakker, Martijn C; Gillespie, Patrick G; McGrouther, Douglas A; Roberts, Anthony H N

2004-12-01

207

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein  

Microsoft Academic Search

BACKGROUND: Lentiviral genomes encode multiple structural and regulatory proteins. Expression of the full complement of viral proteins is accomplished in part by alternative splicing of the genomic RNA. Caprine arthritis encephalitis virus (CAEV) and maedi-visna virus (MVV) are two highly related small-ruminant lentiviruses (SRLVs) that infect goats and sheep. Their genome seems to be less complex than those of primate

Stephen Valas; Morgane Rolland; Cécile Perrin; Gérard Perrin; Robert Z Mamoun

2008-01-01

208

Dissection of splicing regulation at an endogenous locus by zinc-finger nuclease-mediated gene editing.  

PubMed

Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context. PMID:21347446

Cristea, Sandra; Gregory, Philip D; Urnov, Fyodor D; Cost, Gregory J

2011-01-01

209

Dissection of Splicing Regulation at an Endogenous Locus by Zinc-Finger Nuclease-Mediated Gene Editing  

PubMed Central

Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context.

Cristea, Sandra; Gregory, Philip D.; Urnov, Fyodor D.; Cost, Gregory J.

2011-01-01

210

NMR structure of the 5' splice site in the group IIB intron Sc.ai5?--conformational requirements for exon-intron recognition.  

PubMed

A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5' exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5' exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5' exon and the intron. Here, we present the NMR solution structures of the d3' hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg(2+) ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique. PMID:24448450

Kruschel, Daniela; Skilandat, Miriam; Sigel, Roland K O

2014-03-01

211

A randomised controlled pilot study comparing Mepitel(®) and SurfaSoft(®) on paediatric donor sites treated with Recell(®).  

PubMed

This randomized controlled pilot study examined the effects of a silicone net dressing (Mepitel(®)) and a monofilament polyamide woven dressing (SurfaSoft(®)) on the rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal on paediatric donor sites treated with epithelial cell suspension (ReCell(®)). Fifteen children (1-15 years) admitted for acute or reconstructive burns procedures in a tertiary referral hospital in Australia were randomly assigned to the experimental group, Mepitel(®) (n=8) and to the control group, SurfaSoft(®) (n=7). All donor sites were treated with ReCell(®) and covered with the assigned dressing. Measurements of rate of epithelialisation and epidermal maturation, pain, and ease of dressing removal were recorded every two days until the wound was healed. Results showed that there was no difference in the rate of epidermal maturation between the two groups. Less pain and force to remove the dressing was shown in the Mepitel(®) group when compared to SurfaSoft(®). The rate of epithelialisation was found to be an unreliable measure. Although additional research is required to support the results of this study, these results suggest that Mepitel's(®) pliable, self-adhesive and atraumatic properties may improve healing of ReCell(®) treated donor sites with less pain at dressing changes. This pilot study provides a strong base for further research in this area. PMID:21982622

Campanella, S D; Rapley, P; Ramelet, A-S

2011-12-01

212

Evaluation of Human Amniotic Membrane as a Wound Dressing for Split-Thickness Skin-Graft Donor Sites  

PubMed Central

Human amniotic membrane (HAM) has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG) donor sites in a swine model (Part A) and a clinical trial (Part B). Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU) foil (n = 8 each). Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (?SMA: wound contraction marker), von Willebrand factor (vWF: angiogenesis), Ki-67 (cell proliferation), and laminin (basement membrane integrity). Part B: STSG donor sites in 45 adult patients (16 female/29 male) were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n = 15 each). Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative.

Loeffelbein, Denys J.; Rohleder, Nils H.; Eddicks, Matthias; Baumann, Claudia M.; Stoeckelhuber, Mechthild; Wolff, Klaus-D.; Drecoll, Enken; Steinstraesser, Lars; Hennerbichler, Simone; Kesting, Marco R.

2014-01-01

213

Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites.  

PubMed

Human amniotic membrane (HAM) has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG) donor sites in a swine model (Part A) and a clinical trial (Part B). Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU) foil (n = 8 each). Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (?SMA: wound contraction marker), von Willebrand factor (vWF: angiogenesis), Ki-67 (cell proliferation), and laminin (basement membrane integrity). Part B: STSG donor sites in 45 adult patients (16 female/29 male) were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n = 15 each). Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative. PMID:25003117

Loeffelbein, Denys J; Rohleder, Nils H; Eddicks, Matthias; Baumann, Claudia M; Stoeckelhuber, Mechthild; Wolff, Klaus-D; Drecoll, Enken; Steinstraesser, Lars; Hennerbichler, Simone; Kesting, Marco R

2014-01-01

214

Genotype-phenotype associations in neurofibromatosis type 1 (NF1): an increased risk of tumor complications in patients with NF1 splice-site mutations?  

PubMed Central

Neurofibromatosis type 1 (NF1) is a complex neurocutaneous disorder with an increased susceptibility to develop both benign and malignant tumors but with a wide spectrum of inter and intrafamilial clinical variability. The establishment of genotype-phenotype associations in NF1 is potentially useful for targeted therapeutic intervention but has generally been unsuccessful, apart from small subsets of molecularly defined patients. The objective of this study was to evaluate the clinical phenotype associated with the specific types of NF1 mutation in a retrospectively recorded clinical dataset comprising 149 NF1 mutation-known individuals from unrelated families. Each patient was assessed for ten NF1-related clinical features, including the number of café-au-lait spots, cutaneous and subcutaneous neurofibromas and the presence/absence of intertriginous skin freckling, Lisch nodules, plexiform and spinal neurofibromas, optic gliomas, other neoplasms (in particular CNS gliomas, malignant peripheral nerve sheath tumors (MPNSTs), juvenile myelomonocytic leukemia, rhabdomyosarcoma, phaechromocytoma, gastrointestinal stromal tumors, juvenile xanthogranuloma, and lipoma) and evidence of learning difficulties. Gender and age at examination were also recorded. Patients were subcategorized according to their associated NF1 germ line mutations: frame shift deletions (52), splice-site mutations (23), nonsense mutations (36), missense mutations (32) and other types of mutation (6). A significant association was apparent between possession of a splice-site mutation and the presence of brain gliomas and MPNSTs (p?=?0.006). If confirmed, these findings are likely to be clinically important since up to a third of NF1 patients harbor splice-site mutations. A significant influence of gender was also observed on the number of subcutaneous neurofibromas (females, p?=?0.009) and preschool learning difficulties (females, p?=?0.022).

2012-01-01

215

Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5' splice site  

PubMed Central

Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 ( sor1). The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth.

Smalle, Jan A

2013-01-01

216

DNA donors for sequencing at Celera, Craig VenterSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Craig Venter DNAi Location:Genome>the project>players>private project The private project's DNA donors Craig Venter, the leader of the private genome effort at Celera Genomics, talks about the sources of the DNA used in their sequence.

2008-10-06

217

Positive selection acting on splicing motifs reflects compensatory evolution  

PubMed Central

We have used comparative genomics to characterize the evolutionary behavior of predicted splicing regulatory motifs. Using base substitution rates in intronic regions as a calibrator for neutral change, we found a strong avoidance of synonymous substitutions that disrupt predicted exonic splicing enhancers or create predicted exonic splicing silencers. These results attest to the functionality of the hexameric motif set used and suggest that they are subject to purifying selection. We also found that synonymous substitutions in constitutive exons tend to create exonic splicing enhancers and to disrupt exonic splicing silencers, implying positive selection for these splicing promoting events. We present evidence that this positive selection is the result of splicing-positive events compensating for splicing-negative events as well as for mutations that weaken splice-site sequences. Such compensatory events include nonsynonymous mutations, synonymous mutations, and mutations at splice sites. Compensation was also seen from the fact that orthologous exons tend to maintain the same number of predicted splicing motifs. Our data fit a splicing compensation model of exon evolution, in which selection for splicing-positive mutations takes place to counter the effect of an ongoing splicing-negative mutational process, with the exon as a whole being conserved as a unit of splicing. In the course of this analysis, we observed that synonymous positions in general are conserved relative to intronic sequences, suggesting that messenger RNA molecules are rich in sequence information for functions beyond protein coding and splicing.

Ke, Shengdong; Zhang, Xiang H.-F.; Chasin, Lawrence A.

2008-01-01

218

Versatility of retroauricular mastoid donor site: a convenient valuable warehouse of various free graft tissues in cosmetic and reconstructive surgery.  

PubMed

Soft-tissue deficiency is a critical issue in facial cosmetic and reconstructive surgery. Harvesting autografts from other anatomical sites has been a common practice in overcoming soft-tissue insufficiency for many years. However, donor-site complications and visible scars are of important concerns. Therefore, we would like to introduce an alternative donor site of free-tissue grafts and its inherent advantages: the retroauricular mastoid area located along the mastoid hair line. From August 1991 to June 2011, we performed facial reconstructive surgeries for cosmetic correction of disfigurements from both congenital and complications of previous cosmetic procedures on a total of 213 patients. These patients had undergone either 1 or more facial cosmetic surgeries in the past. In this study, our primary goal focused on revising facial asymmetries or defects from previous surgical scars, tissue contraction, undercorrection, or underdevelopment. For autograft harvesting, we incised an elliptical shape along the retroauricular hairline. We then harvested sufficient amount of skin, dermal fat, fascia, or a piece of the mastoid bone if needed. After harvesting, we closed the incisional area and covered it with a compressive dressing. In evaluation of our results, we compared the preoperative photographs with postoperative and constructed a survey on patient satisfaction. Overall, the patients in this study were greatly satisfied with their surgical results. No major complications were reported. As a result of our long-term study, we believe that the retroauricular mastoid area has been shown to be an indispensable donor site for a variety of autograft tissues in terms of safety, convenience, and versatility of its unique structural composition consisting of skin, dermal fat, fascia, and bone. PMID:24036825

Cho, Jeong Mok; Jeong, Jae Hoon; Woo, Kevin Volt; Lee, Yoon Ho

2013-09-01

219

Hydrofiber dressing with silver for the management of split-thickness donor sites: A randomized evaluation of two protocols of care  

Microsoft Academic Search

BackgroundThis randomized, open-label study evaluated Aquacel Ag® Hydrofiber® dressing with silver (HDS; ConvaTec, Skillman, NJ, USA) with an adherent or gelled protocol in the management of split-thickness donor sites.

S. Blome-Eberwein; R. M. Johnson; S. F. Miller; D. M. Caruso; M. H. Jordan; S. Milner; E. E. Tredget; K. M. Sittig; L. Smith

2010-01-01

220

Positive and negative elements mediate control of alternative splicing in the AMPD1 gene.  

PubMed

The second exon of the AMP deaminase (AMPD) 1 gene is alternatively spliced in response to stage-specific signals elaborated during myocyte differentiation. Since inheritance of the mutation in exon 2 of the AMPD1 gene has been recently shown to be associated with a better prognosis of congestive heart failure and the alternative splicing of exon 2 modulates the residual activity of AMPD1 in individuals with this mutant allele, the regulatory mechanism of alternative splicing in the AMPD1 gene is clinically intriguing. Retention or exclusion of exon 2 results from the interplay between negative and positive elements in the primary transcript. Exon 2 is intrinsically defective and difficult to recognize. Herein, we show that this property of exon 2 is the consequence of three defects; a suboptimal 3' splice acceptor site, a suboptimal 5' splice donor site and the small size of the exon. An improvement in any one of these defects relieves the masking of this exon. Further, this defective exon can only be identified in the presence of the adjacent downstream intron. PMID:10767559

Morisaki, H; Morisaki, T; Kariko, K; Genetta, T; Holmes, E W

2000-04-01

221

Immunoglobulin diversification in B cell malignancies: internal splicing of heavy chain variable region as a by-product of somatic hypermutation.  

PubMed

In this study we describe alternative splicing of somatically mutated immunoglobulin (Ig) variable heavy chain (V(H)) genes in three distinct primary B cell non-Hodgkin's lymphomas (B-NHL). In two V4-34 expressing lymphomas, ie a post-germinal center type B cell chronic lymphocytic leukemia (B-CLL) and a follicular lymphoma (FL), internally spliced V(H) gene transcripts were found in which a sequence stretch of 116 bp between the framework region 1 (FR1) and complementarity determining region 2 (CDR2) had been deleted. We provide evidence that for this alternative IgV(H) mRNA processing a known cryptic 5' splice donor site and a previously unidentified cryptic 3' splice acceptor site were used. Site-directed mutagenesis showed that the cryptic 3' splice acceptor site had been activated by specific somatic point mutations. The B-CLL further harbored a triplication of the rearranged JH3 gene segment including the putative N region and part of the JH3-JH4 intron sequence. This triplication probably took place via a repeated mechanism of DNA double strand break followed by homologous recombination, a mechanism which was recently proposed also involved in the somatic hypermutation process and is compatible with the post-germinal center derivation of this B-CLL. Finally, in a V4-34 expressing diffuse large B cell lymphoma, we observed alternative IgV(H) mRNA processing using the same cryptic 5' splice donor site and the normal splice acceptor site of the CH1-C(mu) exon. The significance of alternative IgV(H) processing in B cell malignancies and as a potential mechanism of somatic Ig diversification is discussed. PMID:11960344

Bende, R J; Aarts, W M; Pals, S T; van Noesel, C J M

2002-04-01

222

Donor-site morbidity after pedicled TRAM breast reconstruction: a comparison of two different types of mesh.  

PubMed

Many different approaches have been used to minimize the risk of bulge or hernia formation when using autologous abdominal tissue for breast reconstruction. Studies have shown that further reinforcement of the abdominal wall using a mesh may decrease the complication rate.The current study included 40 consecutive patients having unilateral breast reconstruction with the pedicled transverse rectus abdominus musculocutaneous flap. The defect in the abdominal fascia was closed primarily and further reinforced using a Prolene mesh (Ethicon), n = 20, or using a self-fixating Parietex ProGrip mesh (Covidien), n = 20. The patients were examined at an outpatient consultation, with a minimum follow-up of 1 year and questioned about donor-site symptoms using a standardized questionnaire.Of the 20 patients in the Prolene group, 2 (10%) developed abdominal wall bulging, and of the 20 patients in the ProGrip group, 11 (55%) developed abdominal wall bulging (P = 0.006). In both the Prolene and the ProGrip group, most patients reported having continued donor-site symptoms at the time of the follow-up (70% and 80%, respectively); 15% and 30%, respectively, reported having symptoms that influenced their daily or physical activities (not a significant difference). All but 1 patient in our study reported being very happy with the reconstruction and would have done it again, had they known what they did at the time of the follow-up.We conclude that the self-gripping properties of the Parietex ProGrip mesh are not sufficient in withstanding the abdominal wall tension at the donor site after transverse rectus abdominus musculocutaneous-flap harvest and do not recommend using the Parietex ProGrip mesh without fixating sutures for this procedure. PMID:23392261

Sværdborg, Mille; Damsgaard, Tine Engberg

2013-11-01

223

A new VCAN/versican splice acceptor site mutation in a French Wagner family associated with vascular and inflammatory ocular features  

PubMed Central

Purpose To detail the highly variable ocular phenotypes of a French family affected with an autosomal dominantly inherited vitreoretinopathy and to identify the disease gene. Methods Sixteen family members with ten affected individuals underwent detailed ophthalmic evaluation. Genetic linkage analysis and gene screening were undertaken for genes known to be involved in degenerative and exudative vitreoretinopathies. Qualitative reverse transcriptase-PCR analysis of the versiscan (VCAN) transcripts was performed after mutation detection in the VCAN gene. Results The first index patient of this French family was referred to us because of a chronic uveitis since infancy; this uveitis was associated with exudative retinal detachment in the context of a severe uncharacterized familial vitreoretinopathy. Genetic linkage was obtained to the VCAN locus, and we further identified a new pathogenic mutation at the highly conserved splice acceptor site in intron 7 of the VCAN gene (c.4004–2A>T), which produced aberrantly spliced VCAN transcripts. Conclusions Extensive molecular investigation allowed us to classify this familial vitreoretinopathy as Wagner syndrome. This study illustrates the need to confirm clinical diagnosis by molecular genetic testing and adds new ocular phenotypes to the Wagner syndrome, such as vascular and inflammatory features.

Brezin, Antoine P.; Nedelec, Brigitte; Barjol, Amandine; Rothschild, Pierre-Raphael; Delpech, Marc

2011-01-01

224

Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron.  

PubMed Central

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions. Images

Green, R; Szostak, J W; Benner, S A; Rich, A; Usman, N

1991-01-01

225

A 46,XY Female DSD Patient with Bilateral Gonadoblastoma, a Novel SRY Missense Mutation Combined with a WT1 KTS Splice-Site Mutation  

PubMed Central

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10–15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms’ tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice–site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.

Stoop, Hans; Bernard, Pascal; Sreenivasan, Rajini; Oosterhuis, J. Wolter; Bruggenwirth, Hennie T.; de Boer, Suzan; White, Stefan; Wolffenbuttel, Katja P.; Alders, Marielle; McElreavy, Kenneth; Drop, Stenvert L. S.; Harley, Vincent R.; Looijenga, Leendert H. J.

2012-01-01

226

Alternative splicing of the G protein-coupled receptor superfamily in human airway smooth muscle diversifies the complement of receptors  

PubMed Central

G protein-coupled receptors (GPCRs) are the largest signaling family in the genome, serve an expansive array of functions, and are targets for ?50% of current therapeutics. In many tissues, such as airway smooth muscle (ASM), complex, unexpected, or paradoxical responses to agonists/antagonists occur without known mechanisms. We hypothesized that ASM express many more GPCRs than predicted, and that these undergo substantial alternative splicing, creating a highly diversified receptor milieu. Transcript arrays were designed detecting 434 GPCRs and their predicted splice variants. In this cell type, 353 GPCRs were detected (including 111 orphans), with expression levels varying by ?900-fold. Receptors used for treating airway disease were expressed lower than others with similar signaling properties, indicating potentially more effective targets. A disproportionate number of Class-A peptide-group receptors, and those coupling to Gq/11 or Gs (vs. Gi), was found. Importantly, 192 GPCRs had, on average, five different expressed receptor isoforms because of splicing events, including alternative splice donors and acceptors, novel introns, intron retentions, exon(s) skips, and novel exons, with the latter two events being most prevalent. The consequences of splicing were further investigated with the leukotriene B4 receptor, known for its aberrant responsiveness in lung. We found transcript expression of three variants because of alternative donor and acceptor splice sites, representing in-frame deletions of 38 and 100 aa, with protein expression of all three isoforms. Thus, alternative splicing, subject to conditional, temporal, and cell-type regulation, is a major mechanism that diversifies the GPCR superfamily, creating local recepteromes with specialized environments.

Einstein, Richard; Jordan, Heather; Zhou, Weiyin; Brenner, Michael; Moses, Esther G.; Liggett, Stephen B.

2008-01-01

227

Radiographic evaluation of the symphysis menti as a donor site for an autologous bone graft in pre-implant surgery  

PubMed Central

Purpose This study was performed to obtain a quantitative evaluation of the cortical and cancellous bone graft harvestable from the mental and canine regions, and to evaluate the cortical vestibular thickness. Materials and Methods This study collected cone-beam computed tomographic (CBCT) images of 100 Italian patients. The limits of the mental region were established: 5 mm in front of the medial margin of each mental foramen, 5 mm under the apex of each tooth present, and above the inferior mandibular cortex. Cortical and cancellous bone volumes were evaluated using SimPlant software (SimPlant 3-D Pro, Materialize, Leuven, Belgium) tools. In addition, the cortical vestibular thickness (minimal and maximal values) was evaluated in 3 cross-sections corresponding to the right canine tooth (3R), the median section (M), and the left canine tooth (3L). Results The cortical volume was 0.71±0.23 mL (0.27-1.96 mL) and the cancellous volume was 2.16±0.76 mL (0.86-6.28 mL). The minimal cortical vestibular thickness was 1.54±0.41 mm (0.61-3.25 mm), and the maximal cortical vestibular thickness was 3.14±0.75mm(1.01-5.83 mm). Conclusion The use of the imaging software allowed a patient-specific assessment of mental and canine region bone availability. The proposed evaluation method might help the surgeon in the selection of the donor site by the comparison between bone availability in the donor site and the reconstructive exigency of the recipient site.

Di Bari, Roberto; Coronelli, Roberto

2013-01-01

228

Splicing factor mutations and cancer.  

PubMed

Recent advances in high-throughput sequencing technologies have unexpectedly revealed that somatic mutations of splicing factor genes frequently occurred in several types of hematological malignancies, including myelodysplastic syndromes, other myeloid neoplasms, and chronic lymphocytic leukemia. Splicing factor mutations have also been reported in solid cancers such as breast and pancreatic cancers, uveal melanomas, and lung adenocarcinomas. These mutations were heterozygous and mainly affected U2AF1 (U2AF35), SRSF2 (SC35), SF3B1 (SF3B155 or SAP155), and ZRSR2 (URP), which are engaged in the initial steps of RNA splicing, including 3' splice-site recognition, and occur in a large mutually exclusive pattern, suggesting a common impact of these mutations on RNA splicing. In this study, splicing factor mutations in various types of cancers, their functional/biological effects, and their potential as therapeutic targets have been reviewed. WIREs RNA 2014, 5:445-459. doi: 10.1002/wrna.1222 For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article. PMID:24523246

Yoshida, Kenichi; Ogawa, Seishi

2014-07-01

229

Circulating dendritic cells isolated from healthy seropositive donors are sites of human cytomegalovirus reactivation in vivo.  

PubMed

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34(+) progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation. PMID:23885077

Reeves, Matthew B; Sinclair, John H

2013-10-01

230

Circulating Dendritic Cells Isolated from Healthy Seropositive Donors Are Sites of Human Cytomegalovirus Reactivation In Vivo  

PubMed Central

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34+ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.

Reeves, Matthew B.

2013-01-01

231

Composite tissue transplantation in rats: Fusion of donor muscle to the recipient site  

Microsoft Academic Search

Little information currently exists on the repair of muscular tissue at the site of an amputation stump. This study examined the healing process of muscular tissue following composite limb transplantation using transgenic rat models.

T. Ajiki; A. Kimura; Y. Sato; T. Murakami; Y. Hakamata; Y. Kariya; Y. Hoshino; E. Kobayashi

2005-01-01

232

Entropy Measures Quantify Global Splicing Disorders in Cancer  

PubMed Central

Most mammalian genes are able to express several splice variants in a phenomenon known as alternative splicing. Serious alterations of alternative splicing occur in cancer tissues, leading to expression of multiple aberrant splice forms. Most studies of alternative splicing defects have focused on the identification of cancer-specific splice variants as potential therapeutic targets. Here, we examine instead the bulk of non-specific transcript isoforms and analyze their level of disorder using a measure of uncertainty called Shannon's entropy. We compare isoform expression entropy in normal and cancer tissues from the same anatomical site for different classes of transcript variations: alternative splicing, polyadenylation, and transcription initiation. Whereas alternative initiation and polyadenylation show no significant gain or loss of entropy between normal and cancer tissues, alternative splicing shows highly significant entropy gains for 13 of the 27 cancers studied. This entropy gain is characterized by a flattening in the expression profile of normal isoforms and is correlated to the level of estimated cellular proliferation in the cancer tissue. Interestingly, the genes that present the highest entropy gain are enriched in splicing factors. We provide here the first quantitative estimate of splicing disruption in cancer. The expression of normal splice variants is widely and significantly disrupted in at least half of the cancers studied. We postulate that such splicing disorders may develop in part from splicing alteration in key splice factors, which in turn significantly impact multiple target genes.

Ritchie, William; Granjeaud, Samuel; Puthier, Denis; Gautheret, Daniel

2008-01-01

233

Recurrent mis-splicing of fibrillin exon 32 in two patients with neonatal Marfan syndrome.  

PubMed

The Marfan syndrome (MFS) is an autosomal dominant heritable disorder of connective tissue. Variable and pleiotropic clinical features are observed in the skeletal, ocular, and cardiovascular systems. The most severe end of the phenotypic spectrum of this disorder comprises a group of patients usually diagnosed at birth, who have a life expectancy of little more than a year. To distinguish this group of patients from those with classical MFS, we refer to them as neonatal Marfan syndrome (nMFS). These infants usually die of congestive heart failure rather than aortic aneurysmal disease, the most frequent cause of morbidity and mortality in classical MFS. Defects in fibrillin, an elastin-associated microfibrillar glycoprotein, are now known to cause both the classical and neonatal forms of MFS. Here we report the recurrent mis-splicing of fibrillin (FBN1) exon 32, a precursor EGF-like calcium binding domain, in two unrelated infants with nMFS. The mis-splicing, in one patient, was due to an A-->T transversion at the -2 position of the consensus acceptor splice site; while that in the second patient was caused by a G-->A transition at the +1 position of the donor splice site. Characterization of FBN1 mutations in individuals at the most severe end of the Marfan syndrome spectrum should provide greater understanding of the multiple domains and regions of fibrillin. PMID:7633409

Wang, M; Price, C; Han, J; Cisler, J; Imaizumi, K; Van Thienen, M N; DePaepe, A; Godfrey, M

1995-04-01

234

Psi35 in the branch site recognition region of U2 small nuclear RNA is important for pre-mRNA splicing in Saccharomyces cerevisiae.  

PubMed

Pseudouridine 35 (psi35) in the branch site recognition region of yeast U2 small nuclear RNA is absolutely conserved in all eukaryotes examined. Pus7p catalyzes pseudouridylation at position 35 in Saccharomyces cerevisiae U2. The pus7 deletion strain, although viable in rich medium, is growth-disadvantaged under certain conditions. To clarify the function of U2 psi35 in yeast, we used this pus7 deletion strain to screen a collection of mutant U2 small nuclear RNAs, each containing a point mutation near the branch site recognition sequence, for a synthetic growth defect phenotype. The screen identified two U2 mutants, one containing a U40 --> G40 substitution (U40G) and another having a U40 deletion (U40Delta). Yeast strains carrying either of these U2 mutations grew as well as the wild-type strain in the selection medium, but they exhibited a temperature-sensitive growth defect phenotype when coupled with the pus7 deletion (pus7Delta). A subsequent temperature shift assay and a conditional pus7 depletion (via GAL promoter shutoff) in the U2-U40 mutant genetic background caused pre-mRNA accumulation, suggesting that psi35 is required for pre-mRNA splicing under certain conditions. PMID:15611063

Yang, Chunxing; McPheeters, David S; Yu, Yi-Tao

2005-02-25

235

Where splicing joins chromatin  

PubMed Central

There are numerous data suggesting that two key steps in gene expression—transcription and splicing influence each other closely. For a long time it was known that chromatin modifications regulate transcription, but only recently it was shown that chromatin and histone modifications play a significant role in pre-mRNA splicing. Here we summarize interactions between splicing machinery and chromatin and discuss their potential functional significance. We focus mainly on histone acetylation and methylation and potential mechanisms of their role in splicing. It seems that whereas histone acetylation acts mainly by alterating the transcription rate, histone methylation can also influence splicing directly by recruiting various splicing components.

Hnilicova, Jarmila

2011-01-01

236

Properties of a U1/mRNA 5' splice site duplex containing pseudouridine as measured by thermodynamic and NMR methods.  

PubMed

Three RNA undecamers, 5'AUAC psi psi ACCUG (psi = pseudouridine), 5'AUACUUACCUG, and their complementary 11-mer 5'CAGGUAAGUAU, have been chemically synthesized by phosphite triester chemistry on a controlled-pore glass (CPG) support. The two duplexes formed with these molecules, 5'AUAC psi psi ACCUG/5'CAGGUAAGUAU and 5'AUACUUACCUG/5'CAGGUAAGUAU, represent the 5' end of human U1 snRNA paired to the mRNA consensus 5' splice site. In one undecamer, pseudouridines are incorporated at those positions corresponding to the native in vivo U1 snRNA, while the other (control) undecamer contains only uridine. Surprisingly, the NMR data show that the extra imino proton of the pseudouridines, which is found in the major groove and is presumably not hydrogen bonded, is clearly visible in the imino proton NMR spectrum at pH 6. This result suggests that the structure of the RNA restricts access of solvent to the major groove, slowing the exchange of the pseudouridine NH1 imino proton. A comparison of the thermodynamic properties of the two duplexes show that the free energy of duplex formation is unchanged by the substitution of pseudouridine for uridine. PMID:1993194

Hall, K B; McLaughlin, L W

1991-02-19

237

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties  

Microsoft Academic Search

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US

H. Frances J. Bligh; Patrick D. Larkin; Paul S. Roach; Christopher A. Jones; Hongyong Fu; William D. Park

1998-01-01

238

The protein kinase DYRK1A phosphorylates the splicing factor SF3b1\\/SAP155 at Thr434, a novel in vivo phosphorylation site  

Microsoft Academic Search

BACKGROUND: The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1\\/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells. RESULTS: Overexpression

Katrin de Graaf; Hanna Czajkowska; Sabine Rottmann; Len C Packman; Richard Lilischkis; Bernhard Lüscher; Walter Becker

2006-01-01

239

Premature transcript termination, trans-splicing and DNA repair: a vicious path to cancer  

PubMed Central

So far, about 800 different chromosomal translocations have been characterized in hemato-malignant and solid tumors. Chromosomal translocations mostly result in the expression of chimeric fusion proteins associated with enhanced proliferation and/or malignant transformation. Here, we demonstrate that genes frequently involved in such genetic rearrangements exhibit a unique feature: premature transcriptional termination. These early-terminated RNA molecules have an abundance of 10-20% when compared to their cognate full-length transcripts. They exhibit an unsaturated splice donor site that gives rise to trans-splicing events, leading to RNAs displaying exon repetitions or chimeric fusion RNAs. These arbitrary fusion RNAs mimic the presence of a chromosomal translocation in genetically unaffected cells. Based on our and published data, we propose the hypothesis that these artificial “chimeric fusion transcripts” may influence DNA repair processes, resulting in the generation of de novo chromosomal translocations. This idea provides a rational explanation why different individuals suffer from nearly identical genetic rearrangements.

Kowarz, Eric; Merkens, Jennifer; Karas, Michael; Dingermann, Theo; Marschalek, Rolf

2011-01-01

240

Activated Endothelium Binds Lymphocytes Through a Novel Binding Site in the Alternately Spliced Domain of Vascular Cell Adhesion Molecuh-1  

Microsoft Academic Search

Summary Vascular cell adhesion molecule-1 (VCAM-1) is induced on endothdial cells by inflammatory cytokines, and binds mononuclear leukocytes through the integrin very late antigen-4 (VLA-4) (o14B1). This adhesion pathway has been implicated in a diverse group of physiological and pathological processes, including B cell development, leukocyte activation and recruitment to sites of inflammation, atherosderosis, and tumor cell metastasis. The major

Laurelee Osborn; Cornelia Vassallo; Christopher D. Benjamin

241

Do Functional Keratin Dressings Accelerate Epithelialization in Human Partial Thickness Wounds? A Randomized Controlled Trial on Skin Graft Donor Sites  

PubMed Central

Objective: To determine if the experimental (keratin-based) dressing accelerates epithelialization rates during healing of partial-thickness wounds, relative to a Standard Care dressing. Method: A randomized control trial was conducted using a Standard Care dressing side by side with the experimental dressing on a sample (n=26) of partial-thickness donor site wounds. The proximal/distal placement of the control and treatment was randomized. Percentage epithelialization after approximately 7 days was estimated from which time to fully epithelialize can be inferred. Patients were grouped into “young” (?50 y/o) and “old” (>50 y/o). Results: For the “old” patients (n=15), the median epithelialization percentage at 7 days is 5% and was significantly (P=.023) greater for the experimental dressing. For the “young” patients (n=11), the median epithelialization percentage at 7 days was 80% and there is no significant difference between the experimental and Standard Care control dressings. Conclusions: The experimental dressing significantly increases the rate of epithelialization of acute, traumatic partial-thickness wounds in older patients. We suggest that the dressing may be clinically useful in similar situations where epithelialization may be delayed because of patient or wound characteristics.

Davidson, Andrew; Jina, N. Hamesh; Marsh, Clive; Than, Martin; Simcock, Jeremy W.

2013-01-01

242

[Fillet flaps as a possibility for defect reconstruction of the hand. Reconstruction without additional donor site morbidity].  

PubMed

Fillet flaps offer an additional reconstruction opportunity for complex hand defects after trauma, burns, tumors or infections. This retrospective study elucidates the concept of fillet flaps and presents the results of an overall of 34 plastic surgical reconstructions of the hands in 31 patients. Pedicled axial pattern flaps were used predominantly, except 2 cross finger flaps. In 10 cases the defects were localized in the dorsal and in 9 cases in the palmar aspect of the hand. 14 finger defects and one of the ulnar hand were covered. Very few complications occured. In only 2 cases partial flap loss was observed. An additional wound infection required revision in one case. Another case was left to secondary healing. Prior to any amputation, possible use of spare parts for defect reconstruction should be considered as a matter of principle. Our data suggest that the concept of fillet flaps is suitable for the reconstruction of complex defects of the hands without additional donor site morbidity. PMID:15778828

Noack, N; Hartmann, B; Germann, G; Küntscher, M V

2005-04-01

243

Donor site analgesia after anterior iliac bone grafting in paediatric population: a prospective, triple-blind, randomized clinical trial.  

PubMed

The aim of this study was to compare the efficacy of femoral nerve block with indwelling catheter-based multiple infiltrations of bupivacaine for postoperative pain management after iliac bone harvesting. Sixty paediatric patients undergoing iliac harvesting were randomized into three groups: group A, preoperative femoral nerve block; group B, multiple bolus infiltration of 0.5% bupivacaine via indwelling catheter at the donor site; group C, controls--single dose of 0.5% bupivacaine infiltration given subcutaneously. The primary outcome measure was postoperative pain intensity at rest and at function. The time to maximum pain score, time to ambulation, duration of analgesia, and length of hospital stay were also assessed. Group B patients had the best pain relief and return to function, however the duration of pain relief was longer in group A. Subjects in group A had concomitant motor blockade causing delayed ambulation. Group C showed the worst outcomes. Indwelling catheter-based infiltration of bupivacaine was the most efficient method for providing enhanced pain relief after iliac bone graft harvesting. There was no increase in operating time or hospital stay. Femoral nerve block provided the next best results, but had the significant disadvantage of motor nerve blockade. PMID:24377485

Kumar Raja, D; Anantanarayanan, P; Christabel, A; Manikandhan, R; Elavazhagan, N; Naveen Kumar, J

2014-04-01

244

Conservation and sex-specific splicing of the transformer gene in the calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata.  

PubMed

Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3' end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a "male-only" strain for genetic control programs. PMID:23409170

Li, Fang; Vensko, Steven P; Belikoff, Esther J; Scott, Maxwell J

2013-01-01

245

Conservation and Sex-Specific Splicing of the transformer Gene in the Calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata  

PubMed Central

Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3? end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a “male-only” strain for genetic control programs.

Li, Fang; Vensko, Steven P.; Belikoff, Esther J.; Scott, Maxwell J.

2013-01-01

246

A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs  

PubMed Central

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.

2011-01-01

247

Identification and characterization of a null-activity mutant containing a cryptic pre-mRNA splice site for cytosolic fructose-1,6-bisphosphatase in Flaveria linearis.  

PubMed

Cytosolic fructose-1,6-bisphosphatase (cytFBPase) (E.C. 3.1.3.11) catalyzes the first irreversible reaction of daytime sucrose synthesis. A Flaveria linearis (F. linearis) mutant (line 84-9) previously shown to have ~10% wildtype cytFBPase activity contains no cytFBPase activity based on enzymatic and immunoprecipitation analysis. Genetic segregation and Southern analysis of an F2 population shows one gene copy of cytFBPase in F. linearis and linkage of null cytFBPase activity to the cytFBPase structural gene. A point mutation is present in the structural gene coding for cytFBPase in the mutant, causing a cryptic pre-mRNA splice site and a corresponding 24 amino acid deletion spanning the active site of the enzyme. Collectively, these data support the identification of a null-activity mutant for cytFBPase in F. linearis. This is the first report of a null mutant in the daytime sucrose synthesis pathway confirmed by both enzymatic and molecular analysis. Null cytFBPase in F. linearis does not predispose all lines to high starch accumulation due to an epistatic gene interaction; low starch accumulation in null cytFBPase lines segregates with elevated pyrophosphate-dependent phosphofructokinase (PFP) activity when grown in a 16 h photoperiod. Surprisingly, growth of parental lines and F2 progeny having null cytFBPase in continuous light rescued the wildtype growth phenotype. All null cytFBPase lines showed CO(2)-insensitivity/reversed sensitivity of photosynthesis, indicating that null cytFBPase causes a reduced total capacity for both photosynthesis and end-product synthesis regardless of starch and PFP phenotype. Collectively, the data indicate that F. linearis, a C3-C4 photosynthetic intermediate, has alternative cytFBPase-independent pathways for daytime sucrose synthesis. PMID:20882321

Slater, S M H; Micallef, M C; Zhang, J; Micallef, B J

2010-12-01

248

Defective pre-mRNA splicing in PKD1 due to presumed missense and synonymous mutations causing autosomal dominant polycystic disease.  

PubMed

Autosomal dominant polycystic kidney disease is the most common human monogenic disorder and is caused by mutations in the PKD1 or PKD2 genes. Most patients with the disease present mutations in PKD1, and a considerable number of these alterations are single base substitutions within the coding sequence that are usually predicted to lead to missense or synonymous mutations. There is growing evidence that some of these mutations can be detrimental by affecting the pre-mRNA splicing process. The aim of our study was to test PKD1 mutations, described as missense or synonymous in the literature or databases, for their effects on exon inclusion. Bioinformatics tools were used to select mutations with a potential effect on pre-mRNA splicing. Mutations were experimentally tested using minigene assays. Exons and adjacent intronic sequences were PCR-amplified and cloned in the splicing reporter minigene, and selected mutations were introduced by site-directed mutagenesis. Minigenes were transfected into kidney derived cell lines. RNA from cultured cells was analyzed by RT-PCR and DNA sequencing. Analysis of thirty-three PKD1 exonic mutations revealed three mutations that induce splicing defects. The substitution c.11156G>A, previously predicted as missense mutation p.R3719Q, abolished the donor splice site of intron 38 and resulted in the incorporation of exon 38 with 117bp of intron 38 and skipping of exon 39. Two synonymous variants, c.327A>T (p.G109G) and c.11257C>A (p.R3753R), generated strong donor splice sites within exons 3 and 39 respectively, resulting in incorporation of incomplete exons. These three nucleotide substitutions represent the first PKD1 exonic mutations that induce aberrant mRNAs. Our results strengthen the importance to evaluate the consequences of presumed missense and synonymous mutations at the mRNA level. PMID:24907393

Gonzalez-Paredes, Francisco J; Ramos-Trujillo, Elena; Claverie-Martin, Felix

2014-08-10

249

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

SciTech Connect

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.

Tan, Chye Ling [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Gunaratne, Jayantha [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore)] [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Lai, Deborah [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Carthagena, Laetitia [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France)] [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Wang, Qian [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom)] [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Xue, Yue Zhen; Quek, Ling Shih [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Doorbar, John [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom)] [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Bachelerie, Francoise [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France)] [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Thierry, Francoise, E-mail: francoise.thierry@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Bellanger, Sophie, E-mail: sophie.bellanger@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)] [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)

2012-07-20

250

Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex  

PubMed Central

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (~9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the N? of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and N? in other quinone-processing sites. The experiments with 15N showed that the N? of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the N? of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38 ± 0.13Å. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.

Dikanov, Sergei A.; Holland, J. Todd; Endeward, Burkhard; Kolling, Derrick R. J.; Samoilova, Rimma I.; Prisner, Thomas F.; Antony R., Crofts

2011-01-01

251

Potential therapeutic applications of antisense morpholino oligonucleotides in modulation of splicing in primary immunodeficiency diseases  

Microsoft Academic Search

Highly complementary antisense morpholino oligonucleotides (AMOs) can bind to pre-mRNA and modulate splicing site selection. This offers a powerful tool to regulate the splicing process, such as correcting subtypes of splicing mutations and nonsense mutations and reprogramming alternative splicing processes. Therefore, AMO-mediated splicing modulation represents an attractive therapeutic strategy for genetic disorders. Primary immunodeficiency diseases (PIDs) are a heterogeneous group

Liutao Du; Richard A. Gatti

2011-01-01

252

Elements of the rat tropoelastin gene associated with alternative splicing.  

PubMed

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA. PMID:1572637

Pierce, R A; Alatawi, A; Deak, S B; Boyd, C D

1992-04-01

253

Simian virus 40-rabbit beta-globin recombinants lacking late mRNA splice sites express cytoplasmic RNAs with altered structures.  

PubMed Central

Deletions were introduced at exon-intron boundaries in the late region of a simian virus 40-beta-globin cDNA recombinant to study the role of splicing in the formation of simian virus 40 late cytoplasmic RNAs. The recombinant was used as a wild type because it allowed characterization of mutant RNAs expressed from defective genomes in the presence of comparable RNAs contributed by the coinfecting helper virus. Removal of a 17-base pair segment at map position 0.76, which included a portion of the leader sequence implicated in the splicing of the major 16S mRNA, prevented expression of 16S-type mRNA. The same mutant accumulated cytoplasmic 19S-type mRNA, but the assortment of the 5' ends of these mRNAs differed from the assortment of the wild-type counterparts. Another mutant that lacks nucleotide sequences implicated in the splicing of the major 16S mRNA and one of the principal 19S-type RNAs accumulated a 16S-type mRNA with a previously undetected leader splice, and assortment of 19S mRNAs with new or normally underrepresented splices, and even a species of unspliced cytoplasmic 19S mRNA. Images

White, R T; Berg, P; Villarreal, L P

1982-01-01

254

Genetic analysis of autosomal recessive osteopetrosis in Chuvashiya: the unique splice site mutation in TCIRG1 gene spread by the founder effect.  

PubMed

The rare malignant disorder autosomal recessive osteopetrosis (OPTB) is one of the most prevalent autosomal recessive diseases in the Chuvash Republic of Russia. The purpose of this study was to determine the underlying molecular cause of osteopetrosis in Chuvashiya and to reveal the factors causing the unusual high frequency of the disease in this region. Having assumed a founder effect, we performed linkage disequilibrium (LD) mapping of the OPTB locus at the TCIRG1 region and found a unique splice site mutation c.807+5G>A in all Chuvashian OPTB patients studied. We then analyzed the mutational change in mRNA and detected an intron insertion within the mutant transcript, resulting in a frameshift and premature stop-codon formation (p.Leu271AspfsX231). A decreased expression of the mutant transcript was also detected, which may have been the result of nonsense-mediated decay. Real-time qPCR and MLPA melting curve analysis-based systems were designed and used for c.807+5G>A mutation screening. In addition to analyzing the gene frequency in Chuvashiya, we also estimated three other populations in the Volga-Ural region (Mari, Udmurt and Bashkir). We found a 1.68% prevalence in Chuvashiya (calculated disease frequency, 1/3500 newborns) and a 0.84% in the Mari population (1/14 000 newborns). The haplotype analysis revealed that all OPTB cases in Chuvashians and Marians originated from a single mutational event and the age of the mutation in Chuvashians was estimated to be approximately 890 years. PMID:19172990

Bliznetz, Elena A; Tverskaya, Svetlana M; Zinchenko, Rena A; Abrukova, Anna V; Savaskina, Ekaterina N; Nikulin, Maxim V; Kirillov, Alexander G; Ginter, Evgeny K; Polyakov, Alexander V

2009-05-01

255

A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II.  

PubMed

Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C-->A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II. PMID:19103209

Wang, HaoYang; Hou, YanNing; Cui, YingXia; Huang, YuFeng; Shi, YiChao; Xia, XinYi; Lu, HongYong; Wang, YunHua; Li, XiaoJun

2009-03-01

256

CASE REPORT Persistent Seromas in Abdominal Free Flap Donor Sites After Postmastectomy Breast Reconstruction Surgery: Case Reports and Literature Review  

PubMed Central

Objectives: Donor site seroma formation is a common occurrence following abdominal free flap breast reconstructions. Although such seromas usually resolve spontaneously after a few weeks or months, we recently encountered 3 patients with abdominal seromas persisting for up to 2 years postoperatively. We therefore investigated possible predisposing factors in our patient group. Methods: Patients with persistent abdominal seromas, arbitrarily defined as present after 3 months following abdominal free flap harvest were identified. Their demographic characteristics, comorbidities, reconstruction details, frequency, and volume of abdominal aspirations were documented. Results: Three obese patients (Mean body mass index = 35) with an average age of 49 years bilaterally reconstructed with superior inferior epigastric artery or deep inferior epigastric artery flaps fitted the aforementioned criteria. Seroma aspirations commenced at 3 weeks and continued for a maximum of 26 months postoperatively. The average number of aspirations was 11 with a mean volume of 338 mL (range: 100-864 mL) per visit. The patients were aspirated either weekly or fortnightly depending on the speed of seroma reaccumulation and symptoms. All the 3 patients needed excision of the seroma sac to achieve permanent resolution. Discussion and Conclusion: In addition to their nuisance value (notably frequent aspirations and outpatient clinic visits), persistent seromas can cause significant morbidity and eventually require surgical excision. Possible predisposing factors in our patients included obesity, bilateral reconstructions, and superior inferior epigastric artery flap harvest. Such “high risk” patients should be warned about the likelihood of persistent seromas needing repeated aspirations and possible surgical interventions for ultimate resolution.

Sadeghi, Abtin; Malata, Charles

2013-01-01

257

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

PubMed

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 NpU2AF(65) 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 U2AF(65) 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

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

2014-07-01

258

Splicing analysis of unclassified variants in COL2A1 and COL11A1 identifies deep intronic pathogenic mutations  

PubMed Central

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.

Richards, Allan J; McNinch, Annie; Whittaker, Joanne; Treacy, Becky; Oakhill, Kim; Poulson, Arabella; Snead, Martin P

2012-01-01

259

Anatomical study of the greater palatine artery and related structures of the palatal vault: considerations for palate as the subepithelial connective tissue graft donor site  

Microsoft Academic Search

Palate is considered as a tissue graft donor site for dental surgical procedures. Therefore, the aim of this study was to\\u000a investigate the anatomy of palatal structures, such as greater palatine artery, greater palatine foramen, and incisive fossa,\\u000a in order to consider their topography at planning the graft dimensions and reduce the potential risk of injury of greater\\u000a palatine artery.

Sebastian Krystian Klosek; Thanaporn Rungruang

2009-01-01

260

Indium-labeled white blood cells apheresed from donors receiving G-CSF localize to sites of inflammation when infused into allogeneic bone marrow transplant recipients  

Microsoft Academic Search

G-CSF administration to normal donors results in granulocyte apheresis yields generally greater than those observed with other neutrophil mobilizing agents. In vitro, neutrophils cultured with G-CSF exhibit prolonged survival; however, the random migration of neutrophils exposed to this agent is inhibited. Although transfused neutrophils mobilized with agents other than G-CSF migrate to sites of inflammation or infection in vivo, this

D Adkins; H Goodgold; L Hendershott; M Johnston; D Cravens; G Spitzer

1997-01-01

261

Integrated Study of Ice-Rafted Debris, Temperaturess, and Stable Isotopes on a Spliced Record (piston cores and ODP Site 177-1090) From the South Atlantic  

NASA Astrophysics Data System (ADS)

We have conducted an integrated study of ice-rafted debris (IRD) and stable isotopes on a spliced record (TN057-6-PC4/ODP Site 177-1090, about 43° S, 9° E) raised on the Agulhas Ridge, in the South Atlantic. The site is just north of the northern boundary of the present-day Polar-Front Zone (PFZ), and is in a very sensitive location to record both ice-rafting and stable-isotopic-ratio changes. Our combined record reveals a pattern of ice-rafting episodes that may be characteristic for the subantarctic South Atlantic, at least for locations N of the PFZ. Ice rafting occurs during the waxing stages of each glaciation, and ends at, or before, the peak of each glaciation. IRD peaks are also associated with strong stadials during "cold" interglacials, e.g. MIS 7. A little IRD shows up during the entire interval studied here, from the Holocene to mid-MIS 14. We suggest that the IRD record at this site is essentially a temperature record on glacial-interglacial timescales. If the temperature is low enough, enough icebergs survive to melt at this location. If the temperature is too warm, only an occasional iceberg survives to deliver debris. A peculiar aspect of the combined record is the fact that during Ice-rafting events (IREs), the planktic oxygen-isotopic ratios are higher at the end of an IRE compared to the beginning. Further, by comparing our records with the Summer Sea Surface Temperature record of Becquey and Gersonde (2002) for a nearby (respectedly the same) site (PS2489-2/ODP177-1090), we see that the temperature is generally very similar at the beginning and the end of an IRE. The same age model provided by Venz and Hodell (2002) was used for both sites, allowing such direct comparisons of the data. Assuming as a working hypothesis that the IRD record is a pure temperature record, and ignoring the salinity effect for the present, then this difference in oxygen-isotopic ratios must be the ice volume effect. For MIS 12, the difference in planktic oxygen-isotopic ratios at the beginning and the end of the IRE is about 1.3 permil, which translates to 130 m sea level equivalent. The present-day temperature at the site is about 10° C (Levitus and Boyer, 1998). To attain a temperature of about 4° C (presently located at about 47° S in this area), as indicated by the SSST record of Becquey and Gersonde (2002) for the IRE during MIS 12, the Polar Front Zone (the zone of major iceberg melting) had to move north by about 4° latitude (about 240 nautical miles), a not unreasonable assumption.

Warnke, D. A.; Teitler, L.; Becquey, S.; Gersonde, R.; Venz, K.; Hodell, D. A.

2003-12-01

262

Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript  

PubMed Central

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5'-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

Standiford, D. M.; Davis, M. B.; Sun, W.; Emerson-Jr., C. P.

1997-01-01

263

Electron donor concentrations in sediments and sediment properties at the agricultural chemicals team research site near New Providence, Iowa, 2006-07  

USGS Publications Warehouse

The concentrations of electron donors in aquifer sediments are important to the understanding of the fate and transport of redox-sensitive constituents in groundwater, such as nitrate. For a study by the U.S. Geological Survey National Water-Quality Assessment Program, 50 sediment samples were collected from below the water table from 11 boreholes at the U.S. Geological Survey Agricultural Chemicals Team research site near New Providence, Iowa, during 2006-07. All samples were analyzed for gravel, sand (coarse, medium, and fine), silt, clay, Munsell soil color, inorganic carbon content, and for the following electron donors: organic carbon, ferrous iron, and inorganic sulfide. A subset of 14 sediment samples also was analyzed for organic sulfur, but all of these samples had concentrations less than the method detection limit; therefore, the presence of this potential electron donor was not considered further. X-ray diffraction analyses provided important semi-quantitative information of well-crystallized dominant minerals within the sediments that might be contributing electron donors.

Maharjan, Bijesh; Korom, Scott F.; Smith, Erik A.

2013-01-01

264

The use of moist wound-healing dressings in the management of split-thickness skin graft donor sites: a systematic review.  

PubMed

The aim of this systematic review was to determine the best available evidence related to the post-harvest management of split-thickness skin graft (STSG) donor sites. Studies included in the review were those involving patients of any age examining interventions relating to the post-harvest management of STSG donors and were intra-individual or randomized controlled trials. All studies were checked for methodological quality, and data were extracted using a data extraction tool. Many studies were combined in meta-analysis. The present report concerns studies examining moist and non-moist wound-healing dressings. Broad comparisons of moist wound-healing dressings against traditional non-moist dressings favoured moist wound-healing approaches in terms of healing rates, pain and infection. In comparing dressings within and between moist wound-healing dressing groups, the lack of studies of sufficient quality prevented determining a 'best dressing' for STSG donors. Moist wound-healing products have distinct clinical advantages over non-moist products in the management of STSG donors. There is a strong case for further head-to-head studies comparing products within the moist wound-healing group. PMID:12694482

Wiechula, Rick

2003-04-01

265

The use of Urgotul in the treatment of partial thickness burns and split-thickness skin graft donor sites: a prospective control study.  

PubMed

The use of paraffin-impregnated gauze for burns and skin graft donor sites is commonly associated with wound adherence with consequent pain and trauma upon removal. This prospective clinical study was performed to evaluate a new class of lipido-colloid dressings (Urgotul) in promoting healing and in reducing tissue adherence. In a 6-month period, 25 consecutive patients were recruited. Two separate burn or donor sites on each patient were dressed with tulle-gras (TG) or Urgotul and covered with standard secondary dressings. Objective assessment of wounds by two reviewers, and patients' subjective assessments were recorded. Twenty-three (92%) patients were followed up for a mean of 3 months. Mean time to complete epithelialisation was 9.6 and 11.9 days for the Urgotul and TG sites respectively (P < 0.05). Bleeding was seen in 52% of Urgotul sites compared with 100% of the TG sites at first dressing change (P < 0.05). Patients reported 'moderate pain' during dressing change in 22% and 57% in the Urgotul and TG groups respectively (P < 0.05), with 35% of TG sites being 'very painful' requiring extra analgesia. We found that compared with TG, Urgotul was associated with faster epithelialisation, less pain and trauma (bleeding) during dressing changes. PMID:19719526

Tan, Pearlie W W; Ho, Wong Chin; Song, Colin

2009-08-01

266

Prolyl 4-hydroxylase genes are subjected to alternative splicing in roots of maize seedlings under waterlogging  

PubMed Central

Background In animals, prolyl 4-hydroxylases (P4Hs) are regarded as oxygen sensors under hypoxia stress, but little is known about their role in the response to waterlogging in maize. Methods A comprehensive genome-wide analysis of P4H genes of maize (zmP4H genes) was carried out, including gene structures, phylogeny, protein motifs, chromosomal locations and expression patterns under waterlogging. Key Results Nine zmP4H genes were identified in maize, of which five were alternatively spliced into at least 19 transcripts. Different alternative splicing (AS) events were revealed in different inbred lines, even for the same gene, possibly because of organ and developmental specificities or different stresses. The signal strength of splice sites was strongly correlated with selection of donor and receptor sites, and ambiguous junction sites due to small direct repeats at the exon/intron junction frequently resulted in the selection of unconventional splicing sites. Eleven out of 14 transcripts resulting from AS harboured a premature termination codon, rendering them potential candidates for nonsense-mediated RNA degradation. Reverse transcription–PCR (RT–PCR) indicated that zmP4H genes displayed different expression patterns under waterlogging. The diverse transcripts generated from AS were expressed at different levels, suggesting that zmP4H genes were under specific control by post-transcriptional regulation under waterlogging stress in the line HZ32. Conclusions Our results provide a framework for future dissection of the function of the emerging zmP4H family and suggest that AS might have an important role in the regulation of the expression profile of this gene family under waterlogging stress.

Zou, Xiling; Jiang, Yuanyuan; Zheng, Yonglian; Zhang, Meidong; Zhang, Zuxin

2011-01-01

267

The evolution, impact and properties of exonic splice enhancers  

PubMed Central

Background In humans, much of the information specifying splice sites is not at the splice site. Exonic splice enhancers are one of the principle non-splice site motifs. Four high-throughput studies have provided a compendium of motifs that function as exonic splice enhancers, but only one, RESCUE-ESE, has been generally employed to examine the properties of enhancers. Here we consider these four datasets to ask whether there is any consensus on the properties and impacts of exonic splice enhancers. Results While only about 1% of all the identified hexamer motifs are common to all analyses we can define reasonably sized sets that are found in most datasets. These consensus intersection datasets we presume reflect the true properties of exonic splice enhancers. Given prior evidence for the properties of enhancers and splice-associated mutations, we ask for all datasets whether the exonic splice enhancers considered are purine enriched; enriched near exon boundaries; able to predict trends in relative codon usage; slow evolving at synonymous sites; rare in SNPs; associated with weak splice sites; and enriched near longer introns. While the intersect datasets match expectations, only one original dataset, RESCUE-ESE, does. Unexpectedly, a fully experimental dataset identifies motifs that commonly behave opposite to the consensus, for example, being enriched in exon cores where splice-associated mutations are rare. Conclusions Prior analyses that used the RESCUE-ESE set of hexamers captured the properties of consensus exonic splice enhancers. We estimate that at least 4% of synonymous mutations are deleterious owing to an effect on enhancer functioning.

2013-01-01

268

Novel VCAN mutations and evidence for unbalanced alternative splicing in the pathogenesis of Wagner syndrome  

PubMed Central

Wagner syndrome (WS) is an autosomal dominant vitreoretinopathy affecting various ocular features and is caused by mutations in the canonical splice sites of the VCAN gene, which encodes the large chondroitin sulfate proteoglycan, versican. We report the identification of novel splice acceptor and donor-site mutations (c.4004?1G>C and c.9265+2T>A) in two large WS families from France and the United Kingdom. To characterize their pathogenic mechanisms we performed qRT-PCR experiments on RNA from patient-derived tissues (venous blood and skin fibroblasts). We also analyzed RNA from the original Swiss family reported by Wagner (who has the previously reported c.9265+1G>A mutation). All three mutations resulted in a quantitative increase of transcript variants lacking exons 7 and/or 8. However, the magnitude of the increase varied between tissues and mutations. We discuss altered balance of VCAN splice variants in combination with reduction in glycosaminoglycan protein modifications as possible pathogenic mechanisms.

Kloeckener-Gruissem, Barbara; Neidhardt, John; Magyar, Istvan; Plauchu, Henri; Zech, Jean-Christophe; Morle, Laurette; Palmer-Smith, Sheila M; MacDonald, Moira J; Nas, Veronique; Fry, Andrew E; Berger, Wolfgang

2013-01-01

269

Successful COG8 and PDF overlap is mediated by alterations in splicing and polyadenylation signals.  

PubMed

Although gene-free areas compose the great majority of eukaryotic genomes, a significant fraction of genes overlaps, i.e., unique nucleotide sequences are part of more than one transcription unit. In this work, the evolutionary history and origin of a same-strand gene overlap is dissected through the analysis of COG8 (component of oligomeric Golgi complex 8) and PDF (peptide deformylase). Comparative genomic surveys reveal that the relative locations of these two genes have been changing over the last 445 million years from distinct chromosomal locations in fish to overlapping in rodents and primates, indicating that the overlap between these genes precedes their divergence. The overlap between the two genes was initiated by the gain of a novel splice donor site between the COG8 stop codon and PDF initiation codon. Splicing is accomplished by the use of the PDF acceptor, leading COG8 to share the 3'end with PDF. In primates, loss of the ancestral polyadenylation signal for COG8 makes the overlap between COG8 and PDF mandatory, while in mouse and rat concurrent overlapping and non-overlapping Cog8 transcripts exist. Altogether, we demonstrate that the origin, evolution and preservation of the COG8/PDF same-strand overlap follow similar mechanistic steps as those documented for antisense overlaps where gain and/or loss of splice sites and polyadenylation signals seems to drive the process. PMID:21805148

Pereira-Castro, Isabel; Quental, Rita; da Costa, Luís T; Amorim, António; Azevedo, Luisa

2012-02-01

270

Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms.  

PubMed

The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform. PMID:9499425

Klamt, B; Koziell, A; Poulat, F; Wieacker, P; Scambler, P; Berta, P; Gessler, M

1998-04-01

271

Genomic features defining exonic variants that modulate splicing  

PubMed Central

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.

2010-01-01

272

Detection of human interchromosomal trans-splicing in sequence databanks.  

PubMed

Trans-splicing is a common phenomenon in nematodes and kinetoplastids, and it has also been reported in other organisms, including humans. Up to now, all in silico strategies to find evidence of trans-splicing in humans have required that the candidate sequences follow the consensus splicing site rules (spliceosome-mediated mechanism). However, this criterion is not supported by the best human experimental evidence, which, except in a single case, do not follow canonical splicing sites. Moreover, recent findings describe a novel alternative tRNA mediated trans-splicing mechanism, which prescinds the spliceosome machinery. In order to answer the question, 'Are there hybrid mRNAs in sequence databanks, whose characteristics resemble those of the best human experimental evidence?', we have developed a methodology that successfully identified 16 hybrid mRNAs which might be instances of interchromosomal trans-splicing. Each hybrid mRNA is formed by a trans-spliced region (TSR), which was successfully mapped either onto known genes or onto a human endogenous retrovirus (HERV-K) transcript which supports their transcription. The existence of these hybrid mRNAs indicates that trans-splicing may be more widespread than believed. Furthermore, non-canonical splice site patterns suggest that infrequent splicing sites may occur under special conditions, or that an alternative trans-splicing mechanism is involved. Finally, our candidates are supposedly from normal tissue, and a recent study has reported that trans-splicing may occur not only in malignant tissues, but in normal tissues as well. Our methodology can be applied to 5'-UTR, coding sequences and 3'-UTR in order to find new candidates for a posteriori experimental confirmation. PMID:19955235

Herai, Roberto Hirochi; Yamagishi, Michel E Beleza

2010-03-01

273

A{sup -2} {yields} G transition at the 3{prime} acceptor splice site of IVS17 characterizes the COL2A1 gene mutation in the original Stickler syndrome kindred  

SciTech Connect

Hereditary progressive arthro-ophthalmopathy, or {open_quotes}Stickler syndrome,{close_quotes} is an autosomal dominant osteochondrodysplasia characterized by a variety of ocular and skeletal anomalies which frequently lead to retinal detachment and precocious osteoarthritis. A variety of mutations in the COL2A1 gene have been identified in {open_quotes}Stickler{close_quotes} families; in most cases studied thus far, the consequence of mutation is the premature generation of a stop codon. We report here the characterization of a COL2A1 gene mutation in the original kindred described by Stickler et al. Conformational sensitive gel electrophoresis (CSGE) was used to screen for mutations in the entire COL2A1 gene in an affected member from the kindred. A prominent heteroduplex species was noted in the polymerase chain reaction (PCR) product from a region of the gene including exons 17 to 20. Direct sequencing of PCR-amplified genomic DNA resulted in the identification of a base substitution at the A{sup -2} position of the 3{prime} splice acceptor site of IVS17. Sequencing of DNA from affected and unaffected family members confirmed that the mutation segregated with the disease phenotype. Reverse transcriptase-PCR analysis of poly A+ RNA demonstrated that the mutant allele utilized a cryptic splice site in exon 18 of the gene, eliminating 16 bp at the start of exon 18. This frameshift eventually results in a premature termination codon. These findings are the first report of a splice site mutation in classical Stickler syndrome and they provide a satisfying historical context in which to view COL2A1 mutations in this dysplasia. 25 refs., 3 figs., 1 tab.

Williams, C.J.; Ganguly, A.; Considine, E. [Thomas Jefferson Univ., Philadelphia, PA (United States)] [and others] [Thomas Jefferson Univ., Philadelphia, PA (United States); and others

1996-06-14

274

Pseudouridines in and near the branch site recognition region of U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing in Xenopus oocytes.  

PubMed

Virtually all uridines in the branch site recognition region (BSRR) of vertebrate U2 are converted into pseudouridines after initial transcription. Here, we report a functional analysis of these modified nucleotides using the Xenopus oocyte reconstitution system. Using site-specific (32)P-labeling and TLC, we show that U2 pseudouridylation occurs much faster in the BSRR than in the 5'-terminal region. To functionally dissect the pseudouridines in the BSRR, we replaced each uridine with 5-fluorouridine (unmodifiable nucleotide) using site-specific RNase H cleavage directed by 2'-O-methyl-RNA-DNA chimeras followed by three-piece ligation. Whereas in vitro transcribed U2 containing no 5-fluorouridines rescued splicing in U2-depleted oocytes, no rescue was observed with U2 RNA containing 5-fluorouridines introduced into the BSRR. Additionally, U2 RNA containing 5-fluorouridines in the BSRR specifically inhibited pseudouridylation in the BSRR of in vitro transcribed U2 injected at a later time, although pseudouridylation in the 5'-end region was not affected. Our reconstitution results indicated that prior injection into U2-depleted oocytes with U2 RNA containing 5-fluorouridines in the BSRR almost completely abrogated the ability of in vitro transcribed U2 to rescue splicing, whereas full rescue was obtained with either cellular U2 or U2 containing pseudouridines in the BSRR. Further analyses using glycerol-gradient and native gel electrophoresis indicated that U2 RNAs lacking the BSRR pseudouridines do not participate in the assembly of the functionally active 17S U2 snRNP and the spliceosome. We conclude that the BSRR pseudouridines of vertebrate U2 are required for complete snRNP assembly and pre-mRNA splicing in Xenopus oocytes. PMID:15037777

Zhao, Xinliang; Yu, YiI-Tao

2004-04-01

275

Introduction to Cotranscriptional RNA Splicing  

PubMed Central

The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process.

Merkhofer, Evan C.; Hu, Peter; Johnson, Tracy L.

2014-01-01

276

Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis  

Microsoft Academic Search

Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position 12 (relative to the 5 splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function

Avraham Hury; Hanoch Goldshmidt; Itai Dov Tkacz; Shulamit Michaeli

2009-01-01

277

Randomized trial comparing cryopreserved cultured epidermal allografts with tulle-gras in the treatment of split-thickness skin graft donor sites.  

PubMed

Cultured epidermal allografts have been successfully used to treat a wide variety of skin defects ranging from burns to leg ulcers. Their postulated mechanism of action is through release of multiple cytokines that stimulate epithelialization from the wound periphery as well as from remnant epidermal appendages. A randomized, controlled clinical trial was undertaken to compare the efficacy of cryopreserved cultured allograft dressings (CCAD) with tulle-gras dressings in the treatment of split-skin graft donor sites. Five patients were enrolled in the study and in each patient, half of the donor site was allografted and the other half was treated with tulle-gras control. The mean time to complete healing was 6.2 days for CCAD compared with 9.6 days (p = 0.035) for the tulle-gras controls. Patient assessment of pain with dressing changes was also significantly lower at the CCAD-treated sites than at the control sites (p = 0.001). The results indicate that cultured allografts offer greater patient comfort and earlier maturation of regenerated skin. PMID:8263981

Teepe, R G; Koch, R; Haeseker, B

1993-12-01

278

Splicing shielded cables  

NASA Technical Reports Server (NTRS)

Simple repair technique retains cable characteristic impedance. Shielded-Cable Splicing Technique retains cable characteristic impedance. Procedure involves splicing inner conductors in staggered pattern, installing jumper braid by heat-shrinking two solder sleeves, and placing insulation sleeve over repaired section and heat-shrinking it. Two possible insulation materials are modified polyvinylidene fluoride and polytetrafluoroethylene.

Lind, W. P.; Mcgougan, W. R.

1979-01-01

279

Parallel Arithmetic with Splicing  

Microsoft Academic Search

Abstract. Computing by splicing is one of the main branches of DNA Computing. We address here the problem of computing the basic arithmetical operations in a par- allel way by using splicing systems. Addition, subtraction, multiplication, and integer division between natural numbers are considered. The operations are performed in two steps: the generation of strings and a filtering phase. Both

Pierluigi FRISCO

280

Parallel Arithmetic with Splicing  

Microsoft Academic Search

Computing by splicing is one of the main branches of DNA Computing. We address here the problem of computing the basic arithmetical operations in a par- allel way by using splicing systems. Addition, subtraction, multiplication, and integer division between natural numbers are considered. The operations are performed in two steps: the generation of strings and a filtering phase. Both these

Pierluigi FRISCO

2000-01-01

281

Differential expression of new splice variants of the neurotensin receptor 1 gene in human prostate cancer cell lines.  

PubMed

Neurotensin is a neuroendocrine peptide acting as a trophic factor in a variety of cells in vivo but it can also function as an autocrine growth factor in human prostate cancer cells in vitro. In addition, the high-affinity G protein-coupled NT receptor (NTS1) is overexpressed in prostate cancer cell lines. Increasing evidence argues for a direct correlation between specific alternative splice variants and cancer. We detected four splice variants of the NTS1 receptor in human prostate cancer cell lines. These isoforms include one or more exons skipping as well as an alternative 5' splice donor site and are expressed in the late-stage androgen independent prostate cancer cell lines PC3 and DU145, but not in the early-stage androgen-sensitive LNCaP or in normal prostate tissue, which only express the normal transcript. This result shows new splice variants of NTS1 for the first time. The differential expression observed among prostate cancer cell lines and normal prostate tissue opens the interesting possibility of a new role of NT/NTS1 pathway in prostate cancer. PMID:20018219

Almeida, Teresa A; Rodriguez, Yurena; Hernández, Mariano; Reyes, Ricardo; Bello, Aixa R

2010-02-01

282

Novel and atypical splicing mutation in a compound heterozygous UNC13D defect presenting in Familial Hemophagocytic Lymphohistiocytosis triggered by EBV infection.  

PubMed

Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule priming factor Munc13-4 that intervenes in NK and T cell cytotoxic function. Here we report the case of a 17-month-old girl with prolonged symptomatic EBV infectious mononucleosis and clinical symptoms of hemophagocytic syndrome. In vitro functional analysis pointed to a degranulation defect. The genetic analysis of UNC13D gene identified initially a heterozygous mutation (c.753+1G>T) in the donor splice-site that resulted in exon 9 skipping (maternal allele). Mutations in other genes were considered, but additional analysis of UNC13D cDNA revealed in the paternal allele a heterozygous transition from G to A (c.2448-13G>A) at the 3' acceptor splice-site in intron 25, generating a new acceptor splice-site that leads to a frameshift and a premature STOP codon. Allele specific amplification of the cDNA confirmed the absence of a functional mRNA from the paternal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype. PMID:24825797

Alsina, L; Colobran, R; de Sevilla, M F; Català, A; Viñas, L; Ricart, S; Plaza, A M; Lois, S; Juan, M; Pujol-Borrell, R; Martinez-Gallo, M

2014-08-01

283

Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli.  

PubMed

The selective (15)N isotope labeling was used for the identification of the nitrogen involved in a hydrogen bond formation with the semiquinone in the high-affinity Q(H) site in the cytochrome bo(3) ubiquinol oxidase. This nitrogen produces dominating contribution to X-Band (14)N ESEEM spectra. The 2D ESEEM (HYSCORE) experiments with the Q(H) site SQ in the series of selectively (15)N labeled bo(3) oxidase proteins have directly identified the N(epsilon) of R71 as an H-bond donor. In addition, selective (15)N labeling has allowed us for the first time to determine weak hyperfine couplings with the side-chain nitrogens from all residues around the SQ. Those are reflecting a distribution of the unpaired spin density over the protein in the SQ state of the quinone processing site. PMID:18983149

Lin, Myat T; Samoilova, Rimma I; Gennis, Robert B; Dikanov, Sergei A

2008-11-26

284

Splicing-directed therapy in a new mouse model of human accelerated aging.  

PubMed

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino-based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide-based therapies for treating human diseases of accelerated aging. PMID:22030750

Osorio, Fernando G; Navarro, Claire L; Cadiñanos, Juan; López-Mejía, Isabel C; Quirós, Pedro M; Bartoli, Catherine; Rivera, José; Tazi, Jamal; Guzmán, Gabriela; Varela, Ignacio; Depetris, Danielle; de Carlos, Félix; Cobo, Juan; Andrés, Vicente; De Sandre-Giovannoli, Annachiara; Freije, José M P; Lévy, Nicolas; López-Otín, Carlos

2011-10-26

285

Multiple features contribute to efficient constitutive splicing of an unusually large exon  

PubMed Central

Vertebrate internal exons are usually between 50 and 400 nt long; exons outside this size range may require additional exonic and/or intronic sequences to be spliced into the mature mRNA. The mouse polymeric immunoglobulin receptor gene has a 654 nt exon that is efficiently spliced into the mRNA. We have examined this exon to identify features that contribute to its efficient splicing despite its large size; a large constitutive exon has not been studied previously. We found that a strong 5? splice site is necessary for this exon to be spliced intact, but the splice sites alone were not sufficient to efficiently splice a large exon. At least two exonic sequences and one evolutionarily conserved intronic sequence also contribute to recognition of this exon. However, these elements have redundant activities as they could only be detected in conjunction with other mutations that reduced splicing efficiency. Several mutations activated cryptic 5? splice sites that created smaller exons. Thus, the balance between use of these potential sites and the authentic 5? splice site must be modulated by sequences that repress or enhance use of these sites, respectively. Also, sequences that enhance cryptic splice site use must be absent from this large exon.

Bruce, Shirley R.; Peterson, Martha L.

2001-01-01

286

The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene.  

PubMed Central

The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2-m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5' end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2-m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5' and 3' splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene. Images Fig. 3. Fig. 4. Fig. 6. Fig. 8.

Menssen, A; Hohmann, S; Martin, W; Schnable, P S; Peterson, P A; Saedler, H; Gierl, A

1990-01-01

287

Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5? exons of protein 4.1R gene  

PubMed Central

The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3? splice sites (ss) of exon 2?/2. Here, we demonstrate that exon 1 and 2?/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3? ss and exon 1B to the proximal 3? ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter–driven splicing patterns but differed from 1A-promoter–driven splicing patterns, suggesting that promoter identity affects exon 2?/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2?/2 junction CAGAGAA, a sequence that overlaps the distal U2AF35-binding 3? ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3? ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3? ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network.

Cho, Aeri; Norton, Stephanie; Liu, Eva S.; Park, Jennie; Zhou, Anyu; Munagala, Indira D.; Ou, Alexander C.; Yang, Guang; Wickrema, Amittha; Tang, Tang K.; Benz, Edward J.

2009-01-01

288

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes  

PubMed Central

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.

Juan-Mateu, Jonas; Gonzalez-Quereda, Lidia; Rodriguez, Maria Jose; Verdura, Edgard; Lazaro, Kira; Jou, Cristina; Nascimento, Andres; Jimenez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesus; Baiget, Montserrat; Gallano, Pia

2013-01-01

289

The clinical features of Ehlers-Danlos syndrome type VIIB resulting from a base substitution at the splice acceptor site of intron 5 of the COL1A2 gene.  

PubMed Central

The features of a 32 year old woman with Ehlers-Danlos syndrome type VIIB and affected members of her family, resulting from a mutation in one COL1A2 allele, were studied. Her dermal type I collagen contained alpha 2(I) chains and mutant pN-alpha 2(I) chains in which the amino-terminal propeptide remained attached to the alpha 2(I) chain. She was heterozygous for an AG-->AC mutation at the splice acceptor site of intron 5 of the COL1A2 gene. The mutation activated a cryptic AG splice acceptor site corresponding to positions +14 and +15 of exon 6 of the COL1A2 gene. In contrast to previous reports only five, rather than all 18, amino acids encoded by exon 6 were deleted in the proband. The deleted peptide removed the amino-proteinase cleavage site, but not the nearby lysine cross linking site in the amino-telopeptide of the alpha 2(I) chain. She was born with bilateral hip dislocations, knee subluxations, and generalised joint hypermobility. Bilateral inguinal herniae and an umbilical hernia were present at birth. Facial features included a depressed nasal bridge with prominent paranasal folds. The skin was soft, moderately hyperelastic, and sagged over the face. Skin fragility and easy bruising were apparent from childhood. Skin wounds healed slowly and with broad, paper thin scars. Throughout her life, she had multiple fractures of the small bones of her hands and feet following moderate trauma. Electron microscopy of the proband's dermis as well as deep fascia and hip joint capsule from her affected brother showed that collagen fibrils in transverse section were nearly circular but with irregular margins. Light microscopy of bone from her affected brother and son showed normal Haversian systems and lamellar bone. All of these tissues contained approximately equal amounts of the normal and mutant alpha2(I) chains. The findings of this study confirm that loss of the amino-proteinase cleavage site of the pro alpha2(I) collagen chains, owing to anomalous splicing of exon 6 sequences in the conversion of pre-mRNA to mRNA, produces the clinical features of Ehlers-Danlos syndrome type VIIB. The history of frequent fractures found in this family is atypical and indicates an overlap with osteogenesis imperfecta. Images

Carr, A J; Chiodo, A A; Hilton, J M; Chow, C W; Hockey, A; Cole, W G

1994-01-01

290

SmD1 is required for spliced leader RNA biogenesis.  

PubMed

The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and trans-splicing competence. In Trypanosoma brucei, RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3'-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U28 pseudouridylation was unaffected. PMID:14871954

Zeiner, Gusti M; Foldynová, Silvie; Sturm, Nancy R; Lukes, Julius; Campbell, David A

2004-02-01

291

Poly(ether urethane) wound covering with high water vapour permeability compared with conventional tulle gras on split-skin donor sites.  

PubMed

The experimental poly(ether urethane) (PEU) wound covering with a high water vapour permeance was compared with tulle gras treatment on adjacent areas of the same 20 split-skin donor sites. All patients experienced little or no pain from the PEU-covered areas, while 70 per cent of the patients complained of more pain from the tulle gras-covered areas. The PEU covering did not absorb the wound exudate underneath, neither did it retain wound fluid, but turned the wound exudate into a jelly-like clot layer by allowing a high evaporative water loss from the wound. Tulle gras treatment also prevented wound desiccation, but the exudate was absorbed into the overlaying cotton pads, where it became dry at the outer surface. Microscopy revealed that re-epithelialization occurred at a similar rate under the PEU covering as under tulle gras. In conclusion, the high water vapour permeable PEU wound covering prevents fluid retention, induces clotting of the wound exudate and reduces pain in split-skin donor sites. Tulle gras dressed with gauzes and crêpe bandage prevents wound desiccation, but causes more pain. PMID:2669825

Jonkman, M F; Bruin, P; Pennings, A J; Coenen, J M; Klasen, H J

1989-08-01

292

Feasibility of Use of a Barbed Suture (V-Loc 180) for Quilting the Donor Site in Latissimus Dorsi Myocutaneous Flap Breast Reconstruction  

PubMed Central

Background Latissimus dorsi (LD) myocutaneous flap is a popular method of breast reconstruction which can be associated with high incidence of seroma formation. Quilting sutures at the harvest site are used to reduce this. Barbed sutures are self anchoring sutures which avoid multiple knotting and can be useful in quilting. Methods A retrospective analysis of prospectively maintained database of patients who underwent LD flap breast reconstruction between January 2009 and January 2011 was carried out. Seroma formation at the harvest site, wound related complications, inpatient stay and duration of surgery were analysed and a comparison was made between two groups where quilting was done with barbed (V-Loc) suture and conventional polydioxanone (PDS) II sutures. Results Fifty-seven patients were included of which 33 had quilting by V-Loc sutures and in 24 patients PDS II suture was used. Median age in the PDS group was 55 years (interquartile range [IQR)], 45 to 61 years) which was comparable to the V-Loc group (53 years [IQR, 48 to 59 years]; P-value 0.948). Sixteen patients (28%) had significant seroma formation and 5 (9%) patients developed superficial wound dehiscence. Incidences of seroma or wound complications were comparable (P-value 0.378 and 1.00, respectively). Secondary outcomes such as total duration of surgery, total inpatient stay, total amount of drain at the donor site were also similar in two groups. Conclusions Use of barbed sutures for quilting the donor site in LD flap reconstruction is a feasible option and the associated seroma formation and wound complications are comparable with conventional sutures.

Hussain, Tasadooq; Mahapatra, Tapan Kumar; McManus, Penelope Louise; Kneeshaw, Peter John

2013-01-01

293

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection.  

PubMed

The level of beta-hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2 + 1 G-->A, and a 4-bp deletion, delta CTTT782-785. These mutations, and a 16-kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the delta CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing. PMID:8076944

Kleiman, F E; de Kremer, R D; de Ramirez, A O; Gravel, R A; Argaraña, C E

1994-09-01

294

Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing  

PubMed Central

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 mutations underlying human disease.

Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

2012-01-01

295

A point mutation in an intronic branch site results in aberrant splicing of COL5A1 and in Ehlers-Danlos syndrome type II in two British families.  

PubMed Central

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective-tissue disorders characterized by skin fragility, joint laxity, and skeletal deformities. Type V collagen appears to have a causal role in EDS types I and II, which show phenotypic overlap and may sometimes be allelic. Type V collagen can exist as a heterotrimer, [alpha1(V)]2alpha2(V), and it both coassembles with and regulates type I collagen-fibril diameter. Using an intragenic COL5A1 polymorphism, we have demonstrated linkage, at zero recombination, to the same allele in two large British EDS type II families (LOD scores 4.1 and 4.3). Affected members from each family were heterozygous for a point mutation in intron 32 (IVS32:T-25G), causing the 45-bp exon 33 to be lost from the mRNA in approximately 60% of transcripts from the mutant gene. This mutation lies only 2 bp upstream of a highly conserved adenosine in the consensus branch-site sequence, which is required for lariat formation. Although both families shared the same marker allele, we have been unable to identify a common genealogy. This is the first description of a mutation at the lariat branch site, which plays a pivotal role in the splicing mechanism, in a collagen gene. Very probably, the resulting in-frame exon skip has a dominant-negative effect due to incorporation of the mutant proalpha chain into the triple-helical molecule. These findings further confirm the importance of type V collagen in the causation of EDS type II, and the novel collagen mutation indicates the importance of the lariat branch site in splicing.

Burrows, N P; Nicholls, A C; Richards, A J; Luccarini, C; Harrison, J B; Yates, J R; Pope, F M

1998-01-01

296

An intron 9 containing splice variant of PAX2  

PubMed Central

Background PAX2 is a transcription factor with an important role in embryogenic development. However, PAX2 expression was frequently identified in neoplasia responsible for the growth and survival of cancer cells. Due to alternative splicing of exon 6, exon 10 and exon 12 four isoforms of PAX2 are described so far. Methods The expression of an intron 9 containing PAX2 splice variant was analyzed in neoplastic B cell and solid tumor cell lines as well as in primary tumor samples by quantitative RT-PCR. PAX2 proteins were detected by Western Blot in a subset of cell lines. Results All 14 lymphoma cell lines expressed an undescribed PAX2 splice variant containing the entire intron 9 sequence and the exon 10 sequence. This splice variant could also be detected in 35 solid tumor cell lines, in leukemia and lymphoma as well as in colon carcinoma and melanoma patient samples and in blood samples of healthy donors. Expression of this new splice variant on protein level was verified by Western Blot analysis. Conclusion We discovered a previously undescribed intron 9 and exon 10 containing splice variant of PAX2 in B-cell neoplasia and in solid tumors on mRNA and protein level.

Busse, Antonia; Rietz, Anika; Schwartz, Stefan; Thiel, Eckhard; Keilholz, Ulrich

2009-01-01

297

Some relations between two stages DNA splicing languages  

NASA Astrophysics Data System (ADS)

A new symbolization of Yusof-Goode (Y-G) rule, which is associated with Y-G splicing system, was introduced by Yusof in 2012 under the framework of formal language theory. The purpose of this investigation is to present the biological process of DNA splicing in a translucent way. In this study, two stages splicing languages are introduced based on Y-G approach and some relations between stage one and stage two splicing languages are presented, given as theorems. Additionally, the existing relations between two stages splicing languages based on crossings and contexts of restriction enzymes factors with respect to two initial strings (having two cutting sites) and two rules are presented as subset.

Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

2014-06-01

298

Copper-Sulfur Complexes Supported by N-Donor Ligands: Towards Models of the CuZ Site in Nitrous Oxide Reductase  

PubMed Central

The distinctive structure of the [(his)7Cu4(?-S)]n+ cluster in the “CuZ” active site of nitrous oxide reductase and the intriguing mechanistic hypotheses for its catalytic reactivity provide inspiration for synthetic model studies aimed at characterizing relevant copper-sulfur compounds and obtaining fundamental insights into structure and bonding. In this brief review, we summarize such studies that have focused on the synthesis and characterization of a range of copper-sulfur complexes supported by N-donor ligands. Compounds with variable nuclearities and sulfur redox levels have been isolated, with the nature of the species obtained being dependent on the supporting ligand, sulfur source, and the reaction conditions. Spectroscopic data and theoretical calculations, often performed with a view toward drawing comparisons to oxygen analogs, have provided insight into the nature of the copper-sulfur bonding interactions in the complexes.

York, John T.; Bar-Nahum, Itsik; Tolman, William B.

2008-01-01

299

Splicing regulation as a potential genetic modifier  

Microsoft Academic Search

Inherited diseases are associated with profound phenotypic variability, which is affected strongly by genetic modifiers. The splicing machinery could be one such modifying system, through a mechanism involving splicing motifs and their interaction with a complex repertoire of splicing factors. Mutations in splicing motifs and changes in levels of splicing factors can result in different splicing patterns. Changes in the

Malka Nissim-Rafinia; Batsheva Kerem

2002-01-01

300

Insertion of non-intron sequence into maize introns interferes with splicing.  

PubMed Central

Transposable element (TE) insertion into or near plant introns can cause intron skipping and alternative splicing events, resulting in reduced expression. To explore the impact of inserted sequences on splicing, we added non-intron sequence to two maize introns and tested these chimeric introns in a maize transient expression assay. Non-intron sequence inserted into Adh1-S intron 1 and actin intron 3 decreased expression from the luciferase reporter gene; the insertion sites tested were not in intron regions thought to be essential for splicing. Alternatively spliced mRNAs were not observed in transcripts derived from the insertion variants. In contrast, addition of an internal segment of an intron to Adh1-S intron 1 resulted in normal splice site selection and efficient processing. Because the normal intron sequence (including the conserved splice junctions) was retained in all constructs, we hypothesize that added non-intron sequence can interfere with intron recognition and/or splicing. Images

Luehrsen, K R; Walbot, V

1992-01-01

301

Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery  

PubMed Central

The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice. At the molecular level, the absence of PRMT5 results in reduced methylation of Sm proteins, aberrant constitutive splicing, and the alternative splicing of specific mRNAs with weak 5? donor sites. Intriguingly, the products of these mRNAs are, among others, several proteins regulating cell cycle progression. We identify Mdm4 as one of these key mRNAs that senses the defects in the spliceosomal machinery and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in vivo. Our data demonstrate that PRMT5 is a master regulator of splicing in mammals and uncover a new role for the Mdm4 pre-mRNA, which could be exploited for anti-cancer therapy.

Bezzi, Marco; Teo, Shun Xie; Muller, Julius; Mok, Wei Chuen; Sahu, Sanjeeb Kumar; Vardy, Leah A.; Bonday, Zahid Q.; Guccione, Ernesto

2013-01-01

302

Gene Therapeutic Approach Using Mutation-adapted U1 snRNA to Correct a RPGR Splice Defect in Patient-derived Cells  

PubMed Central

Retinitis pigmentosa (RP) is a disease that primarily affects the peripheral retina and ultimately causes visual impairment. X-chromosomal forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We show that the novel splice donor site (SDS) mutation c.1245+3A>T in intron 10 of RPGR cosegregates with RP in a five-generation Caucasian family. The mutation causes in-frame skipping of exon 10 from RPGR transcripts in patient-derived primary fibroblasts. To correct the splice defect, we developed a gene therapeutic approach using mutation-adapted U1 small nuclear RNA (U1). U1 is required for SDS recognition of pre-mRNAs and initiates the splice process. The mutation described herein interferes with the recognition of the SDS by U1. To overcome the deleterious effects of the mutation, we generated four U1 isoforms with increasing complementarity to the SDS. Lentiviral particles were used to transduce patient-derived fibroblasts with these U1 variants. Full complementarity of U1 corrects the splice defect partially and increases recognition of the mutant SDS. The therapeutic effect is U1-concentration dependent as we show for endogenously expressed RPGR transcripts in patient-derived cells. U1-based gene therapeutic approaches constitute promising technologies to treat SDS mutations in inherited diseases including X-linked RP.

Glaus, Esther; Schmid, Fabian; Da Costa, Romain; Berger, Wolfgang; Neidhardt, John

2011-01-01

303

More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns.  

PubMed

Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA. PMID:9769094

Chu, V T; Liu, Q; Podar, M; Perlman, P S; Pyle, A M

1998-10-01

304

U1 Small Nuclear Ribonucleoprotein and Splicing Inhibition by the Rous Sarcoma Virus Negative Regulator of Splicing Element  

PubMed Central

Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5? splice site (5? ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3? ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5? half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.

McNally, Lisa M.; McNally, Mark T.

1999-01-01

305

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B  

PubMed Central

Modulation of alternative pre-mRNA splicing is a potential approach to therapeutic targeting for a variety of human diseases. We investigated the mechanism by which digitoxin, a member of the cardiotonic steroid class of drugs, regulates alternative splicing. Transcriptome-wide analysis identified a large set of alternative splicing events that change after digitoxin treatment. Within and adjacent to these regulated exons, we identified enrichment of potential binding sites for the splicing factors SRp20 (SRSF3/SFRS3) and Tra2-? (SFRS10/TRA2B). We further find that both of these proteins are depleted from cells by digitoxin treatment. Characterization of SRp20 and Tra2-? splicing targets revealed that many, but not all, digitoxin-induced splicing changes can be attributed to the depletion of one or both of these factors. Re-expression of SRp20 or Tra2-? after digitoxin treatment restores normal splicing of their targets, indicating that the digitoxin effect is directly due to these factors. These results demonstrate that cardiotonic steroids, long prescribed in the clinical treatment of heart failure, have broad effects on the cellular transcriptome through these and likely other RNA binding proteins. The approach described here can be used to identify targets of other potential therapeutics that act as alternative splicing modulators.

Anderson, Erik S.; Lin, Chia-Ho; Xiao, Xinshu; Stoilov, Peter; Burge, Christopher B.; Black, Douglas L.

2012-01-01

306

Tissue-Specific Expression of a Splicing Mutation in the IKBKAP Gene Causes Familial Dysautonomia  

PubMed Central

Familial dysautonomia (FD; also known as “Riley-Day syndrome”), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

Slaugenhaupt, Susan A.; Blumenfeld, Anat; Gill, Sandra P.; Leyne, Maire; Mull, James; Cuajungco, Math P.; Liebert, Christopher B.; Chadwick, Brian; Idelson, Maria; Reznik, Luba; Robbins, Christiane M.; Makalowska, Izabela; Brownstein, Michael J.; Krappmann, Daniel; Scheidereit, Claus; Maayan, Channa; Axelrod, Felicia B.; Gusella, James F.

2001-01-01

307

Two Tunisian patients with Peters plus syndrome harbouring a novel splice site mutation in the B3GALTL gene that modulates the mRNA secondary structure.  

PubMed

Peters plus syndrome is an autosomal recessive rare disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities, distinctive facial features, and often other major/minor additional defects. Peters plus syndrome is related to mutations in the B3GALTL gene with only seven recently reported mutations, leading to the inactivation of the B1, 3-glucosyltransferase. In this study, we screened the B3GALTL gene in two unrelated patients with typical Peters plus syndrome. A novel homozygous c.597-2A>G mutation was identified in both patients. Bioinformatic analyses showed that this mutation modulates the pre mRNA secondary structure of the gene, and decreases the score value related to the formation of splicing loops. Moreover, the c.597-2A>G mutation is located in a CpG Island of the B3GALTL gene, suggesting a potential epigenetic role of this position including gene's methylation and regulation. These data confirm an important role of the B3GALTL gene test that provides diagnosis confirmation and improves genetic counseling for the families. PMID:22759511

Siala, Olfa; Belguith, Neila; Kammoun, Hassen; Kammoun, Bourane; Hmida, Nedia; Chabchoub, Imen; Hchicha, Mongia; Fakhfakh, Faiza

2012-10-01

308

Retroperitoneal LESS donor nephrectomy.  

PubMed

Donor nephrectomy with laparo-endoscopic single site (LESS) surgery has been reported via the transperitoneal approach. We describe a novel technique of retroperitoneal donor nephrectomy using a single surgical incision in the groin, below the abdominal skin crease or "bikini line". The LESS groin incision offers superior cosmesis, while the retroperitoneal approach has distinct advantages, such as the ability to identify the renal vessels early. The new procedure has been performed in two obese patients (body mass index 32 and 33 kg/m2, respectively). The operative times were 4 and 5 hours, warm ischemic times 135 and 315 seconds, blood loss 100 and 250 mL, and hospitalization 3 and 2 days, respectively. Retroperitoneal LESS donor nephrectomy through a single, inconspicuous groin incision is feasible and safe. Further evaluation of the technique in a larger patient cohort is indicated. PMID:21044377

van der Merwe, A; Bachmann, A; Heyns, C F

2010-01-01

309

The transcription factor c-Myb affects pre-mRNA splicing  

SciTech Connect

c-Myb is a transcription factor which plays a key role in haematopoietic proliferation and lineage commitment. We raised the question of whether c-Myb may have abilities beyond the extensively studied transcriptional activation function. In this report we show that c-Myb influences alternative pre-mRNA splicing. This was seen by its marked effect on the 5'-splice site selection during E1A alternative splicing, while no effect of c-Myb was observed when reporters for the 3'-splice site selection or for the constitutive splicing process were tested. Moreover, co-immunoprecipitation experiments provided evidence for interactions between c-Myb and distinct components of the splicing apparatus, such as the general splicing factor U2AF{sup 65} and hnRNPA1 involved in the 5'-splice site selection. The effect on 5'-splice site selection was abolished in the oncogenic variant v-Myb. Altogether, these data provide evidence that c-Myb may serve a previously unappreciated role in the coupling between transcription and splicing.

Orvain, Christophe; Matre, Vilborg [University of Oslo, Department of Molecular Biosciences, P.O. Box 1041 Blindern, N-0316 Oslo (Norway); Gabrielsen, Odd S. [University of Oslo, Department of Molecular Biosciences, P.O. Box 1041 Blindern, N-0316 Oslo (Norway)], E-mail: o.s.gabrielsen@imbv.uio.no

2008-07-25

310

Safer, Silencing-Resistant Lentiviral Vectors: Optimization of the Ubiquitous Chromatin-Opening Element through Elimination of Aberrant Splicing  

PubMed Central

Gammaretroviral and lentiviral vectors have been used successfully in several clinical gene therapy trials, although powerful enhancer elements have caused insertional mutagenesis and clonal dysregulation. Self-inactivating vectors with internal heterologous regulatory elements have been developed as potentially safer and more effective alternatives. Lentiviral vectors containing a ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 locus (A2UCOE), which allows position-independent, long-term transgene expression, are particularly promising. In a recently described assay, aberrantly spliced mRNA transcripts initiated in the vector A2UCOE sequence were found to lead to upregulation of growth hormone receptor gene (Ghr) expression in transduced murine Bcl-15 cells. Aberrant hybrid mRNA species formed between A2UCOE and a number of other cellular genes were also detected in transduced human PLB-985 myelomonocytic cells. Modification of the A2UCOE by mutation or deletion of recognized and potential cryptic splice donor sites was able to abrogate these splicing events and hybrid mRNA formation in Bcl-15 cells. This modification did not compromise A2UCOE regulatory activity in terms of resistance to CpG methylation and gene silencing in murine P19 embryonic carcinoma cells. These refined A2UCOE regulatory elements are likely to improve intrinsic biosafety and may be particularly useful for a number of clinical applications where robust gene expression is desirable.

Knight, Sean; Zhang, Fang; Mueller-Kuller, Uta; Bokhoven, Marieke; Gupta, Abhinav; Broughton, Thomas; Sha, Sha; Antoniou, Michael N.; Brendel, Christian; Grez, Manuel; Thrasher, Adrian J.; Collins, Mary

2012-01-01

311

Complicated RNA splicing of chicken telomerase reverse transcriptase revealed by profiling cells both positive and negative for telomerase activity.  

PubMed

Telomerase reverse transcriptase (TERT) is an essential component of the telomerase ribonucleoprotein complex which maintains telomeres. The objective of this study was to investigate chicken TERT (cTERT) alternative RNA splicing profiles of samples varying for telomerase activity and immortalization parameters. These included systems both in vivo (gastrula embryo, embryo and adult liver) and in vitro (chicken embryo fibroblasts (CEFs) and DT40 cells). Nineteen cTERT variants were discovered, which were generated through exon skipping, intron retention, and alternative usage of splice donor and acceptor sites. Three variants were predicted to introduce in-frame mutations, whereas the others were predicted to have premature termination codons. The number of cTERT variants detected ranged from 10 in adult liver to 13 in CEFs. One variant (V4) was found in all samples and was predicted to generate a truncated protein lacking telomerase catalytic activity. Interestingly, the standard TERT expected from the full-length transcript was expressed not only in telomerase-positive, but also in telomerase-negative samples. The complicated expression profiles of cTERT in various cell systems suggest that sophisticated regulatory pathways are involved in cTERT pre-mRNA editing. Further, these results support the body of increasing evidence that alternative splicing of TERT, both in human and chicken, contributes to telomerase activity regulation. PMID:16806743

Chang, Hong; Delany, Mary E

2006-09-01

312

FOA Lecture 6: Fiber Optic Splices  

NSDL National Science Digital Library

This is the sixth lecture in the FOA series on fiber optics, covering splices. In this lecture the presenter discusses the uses of splices, types of splices such as fusion splices and mechanical splices, and the processes used for making each type of splice. The video also discusses cleaving, which is the key to getting good splices. Running time for the lecture is 9:03. Flash is required to view the video.

2013-07-08

313

Hydrogen bonds between nitrogen donors and the semiquinone in the QB-site of bacterial reaction centers  

PubMed Central

Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone-10 molecules functioning as primary (QA) and secondary (QB) electron acceptors. X-band 2D pulsed EPR spectroscopy, called HYSCORE, was applied to study the interaction of the QB site semiquinone with nitrogens from the local protein environment in natural and 15N uniformly labeled reactions centers. 14N and 15N HYSCORE spectra of the QB semiquinone show the interaction with two nitrogens carrying transferred unpaired spin density. Quadrupole coupling constants estimated from 14N HYSCORE spectra indicate them to be a protonated nitrogen of an imidazole residue and amide nitrogen of a peptide group. 15N HYSCORE spectra allowed estimation of the isotropic and anisotropic couplings with these nitrogens. From these data, we calculated the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen, and analyzed the contribution of different factors to the anisotropic hyperfine tensors. The hyperfine coupling of other protein nitrogens with the semiquinone is weak (<0.1 MHz). These results clearly indicate that the QB semiquinone forms hydrogen bonds with two nitrogens, and provide quantitative characteristics of the hyperfine couplings with these nitrogens, which can be used in theoretical modeling of the QB site. Based on the quadrupole coupling constant, one nitrogen can only be assigned to N? of His-L190, consistent with all existing structures. However, we cannot specify between two candidates the residue corresponding to the second nitrogen. Further work employing multifrequency spectroscopic approaches or selective isotope labeling would be desirable for unambiguous assignment of this nitrogen.

Martin, Erik; Samoilova, Rimma I.; Narasimhulu, Kupala V.; Wraight, Colin A.; Dikanov, Sergei A.

2010-01-01

314

Hydrogen bonds between nitrogen donors and the semiquinone in the Q(B) site of bacterial reaction centers.  

PubMed

Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone-10 molecules functioning as primary (Q(A)) and secondary (Q(B)) electron acceptors. X-band 2D pulsed EPR spectroscopy, called HYSCORE, was applied to study the interaction of the Q(B) site semiquinone with nitrogens from the local protein environment in natural and (15)N uniformly labeled reactions centers. (14)N and (15)N HYSCORE spectra of the Q(B) semiquinone show the interaction with two nitrogens carrying transferred unpaired spin density. Quadrupole coupling constants estimated from (14)N HYSCORE spectra indicate them to be a protonated nitrogen of an imidazole residue and amide nitrogen of a peptide group. (15)N HYSCORE spectra allowed estimation of the isotropic and anisotropic couplings with these nitrogens. From these data, we calculated the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and analyzed the contribution of different factors to the anisotropic hyperfine tensors. The hyperfine coupling of other protein nitrogens with the semiquinone is weak (<0.1 MHz). These results clearly indicate that the Q(B) semiquinone forms hydrogen bonds with two nitrogens and provide quantitative characteristics of the hyperfine couplings with these nitrogens, which can be used in theoretical modeling of the Q(B) site. On the basis of the quadrupole coupling constant, one nitrogen can only be assigned to N(delta) of His-L190, consistent with all existing structures. However, we cannot specify between two candidates the residue corresponding to the second nitrogen. Further work employing multifrequency spectroscopic approaches or selective isotope labeling would be desirable for unambiguous assignment of this nitrogen. PMID:20672818

Martin, Erik; Samoilova, Rimma I; Narasimhulu, Kupala V; Wraight, Colin A; Dikanov, Sergei A

2010-08-25

315

Splicing-Related Features of Introns Serve to Propel Evolution  

PubMed Central

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

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

2013-01-01

316

Site Saturation Mutagenesis Demonstrates a Central Role for Cysteine 298 as Proton Donor to the Catalytic Site in CaHydA [FeFe]-Hydrogenase  

PubMed Central

[FeFe]-hydrogenases reversibly catalyse molecular hydrogen evolution by reduction of two protons. Proton supply to the catalytic site (H-cluster) is essential for enzymatic activity. Cysteine 298 is a highly conserved residue in all [FeFe]-hydrogenases; moreover C298 is structurally very close to the H-cluster and it is important for hydrogenase activity. Here, the function of C298 in catalysis was investigated in detail by means of site saturation mutagenesis, simultaneously studying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstrated that efficient enzymatic turnover was maintained only when C298 was replaced by aspartic acid, despite the structural diversity between the two residues. Purified CaHydA C298D does not show any significant structural difference in terms of secondary structure and iron incorporation, demonstrating that the mutation does not affect the overall protein fold. C298D retains the hydrogen evolution activity with a decrease of kcat only by 2-fold at pH 8.0 and it caused a shift of the optimum pH from 8.0 to 7.0. Moreover, the oxygen inactivation rate was not affected demonstrating that the mutation does not influence O2 diffusion to the active site or its reactivity with the H-cluster. Our results clearly demonstrate that, in order to maintain the catalytic efficiency and the high turnover number typical of [FeFe] hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvement in the catalytic function of [FeFe]-hydrogenases in agreement with an essential role in proton transfer to the active site.

Morra, Simone; Giraudo, Alberto; Di Nardo, Giovanna; King, Paul W.; Gilardi, Gianfranco; Valetti, Francesca

2012-01-01

317

Donor Tag Game  

MedlinePLUS

... Games > Donor Tag Game Printable Version Donor Tag Game This feature requires version 6 or later of ... Needles Blood Donor Community Donor Stories Recipient Stories Games Facebook Fanbox Avatars and Badges Banners eCards Enter ...

318

Design principles for bifunctional targeted oligonucleotide enhancers of splicing.  

PubMed

Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a 'tail' of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns. PMID:21602265

Owen, Nicholas; Zhou, Haiyan; Malygin, Alexey A; Sangha, Jason; Smith, Lindsay D; Muntoni, Francesco; Eperon, Ian C

2011-09-01

319

Genome-wide survey of Alternative Splicing in Sorghum Bicolor.  

PubMed

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

Panahi, Bahman; Abbaszadeh, Bahram; Taghizadeghan, Mehdi; Ebrahimie, Esmaeil

2014-07-01

320

Recovery and safety profiles of marrow and PBSC donors: experience of the National Marrow Donor Program.  

PubMed

The National Marrow Donor Program (NMDP) has been facilitating hematopoietic cell transplants since 1987. Volunteer donors listed on the NMDP Registry may be asked to donate either bone marrow (BM) or peripheral blood stem cells (PBSC); however, since 2003, the majority of donors (72% in 2007) have been asked to donate PBSC. From the donor's perspective these stem cell sources carry different recovery and safety profiles. The majority of BM and PBSC donors experienced symptoms during the course of their donation experience. Pain is the number 1 symptom for both groups of donors. BM donors most often reported pain at the collection site (82% back or hip pain) and anesthesia-related pain sites (33% throat pain; 17% post-anesthesia headache), whereas PBSC donors most often reported bone pain (97%) at various sites during filgrastim administration. Fatigue was the second most reported symptom by both BM and PBSC donors (59% and 70%, respectively). PBSC donors reported a median time to recovery of 1 week compared to a median time to recovery of 3 weeks for BM donors. Both BM and PBSC donors experienced transient changes in their WBC, platelet, and hemoglobin counts during the donation process, with most counts returning to baseline values by 1 month post-donation and beyond. Serious adverse events are uncommon, but these events occurred more often in BM donors than PBSC donors (1.34% in BM donors, 0.6% in PBSC donors) and a few BM donors may have long-term complications. NMDP donors are currently participating in a randomized clinical trial that will formally compare the clinical and quality-of-life outcomes of BM and PBSC donors and their graft recipients. PMID:18721778

Miller, John P; Perry, Elizabeth H; Price, Thomas H; Bolan, Charles D; Karanes, Chatchada; Boyd, Theresa M; Chitphakdithai, Pintip; King, Roberta J

2008-09-01

321

Anatomical study of the greater palatine artery and related structures of the palatal vault: considerations for palate as the subepithelial connective tissue graft donor site.  

PubMed

Palate is considered as a tissue graft donor site for dental surgical procedures. Therefore, the aim of this study was to investigate the anatomy of palatal structures, such as greater palatine artery, greater palatine foramen, and incisive fossa, in order to consider their topography at planning the graft dimensions and reduce the potential risk of injury of greater palatine artery. Direct inspection of 41 Thai cadavers was performed. The results showed the statistically significant differences as for the length of female and male palates (p = 0.017); however, vertical measurements were equally distributed in examined population. Main location of greater palatine foramen was palatal to the second molar (35.7%), as well as, interproximal to the second and third molars (35.7%) in women, and palatal to the second molar in men (65%). GPA was branching most frequently at the level of first premolar (38%) and at first and second molars together (43%) in women. In men, the branching on the alveolar process side was commonly observed at the level of first and second premolars together (56%), and at the level of second and third molars together (32%). In the area between maxillary first premolar and second molar, it appeared possible to harvest a connective tissue graft measuring at least 5 mm in height. The results of this research will provide the useful data for other comparative studies and for assisting periodontologists in planning the dimensions and harvesting the subepithelial connective tissue grafts from palate. PMID:19015806

Klosek, Sebastian Krystian; Rungruang, Thanaporn

2009-04-01

322

Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo  

PubMed Central

In vivodelivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD-scid IL2r?null mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA, and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen, and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the ?-globin gene using nanoparticles carrying ?-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an EGFP- ?-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

McNeer, Nicole A.; Schleifman, Erica B.; Cuthbert, Amy; Brehm, Michael; Jackson, Andrew; Cheng, Christopher; Anandalingam, Kavitha; Kumar, Priti; Shultz, Leonard D.; Greiner, Dale L.

2013-01-01

323

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

PubMed

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

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

324

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

PubMed Central

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.

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

2008-01-01

325

Splicing Wires Permanently With Explosives  

NASA Technical Reports Server (NTRS)

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.

Bement, Laurence J.; Kushnick, Anne C.

1990-01-01

326

Nested introns in an intron: evidence of multi-step splicing in a large intron of the human dystrophin pre-mRNA.  

PubMed

The mechanisms by which huge human introns are spliced out precisely are poorly understood. We analyzed large intron 7 (110199 nucleotides) generated from the human dystrophin (DMD) pre-mRNA by RT-PCR. We identified branching between the authentic 5' splice site and the branch point; however, the sequences far from the branch site were not detectable. This RT-PCR product was resistant to exoribonuclease (RNase R) digestion, suggesting that the detected lariat intron has a closed loop structure but contains gaps in its sequence. Transient and concomitant generation of at least two branched fragments from nested introns within large intron 7 suggests internal nested splicing events before the ultimate splicing at the authentic 5' and 3' splice sites. Nested splicing events, which bring the authentic 5' and 3' splice sites into close proximity, could be one of the splicing mechanisms for the extremely large introns. PMID:23395799

Suzuki, Hitoshi; Kameyama, Toshiki; Ohe, Kenji; Tsukahara, Toshifumi; Mayeda, Akila

2013-03-18

327

A novel approach to describe a U1 snRNA binding site  

PubMed Central

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5? end of U1 snRNA and 5? splice sites and numerous mutations following transient transfection of HeLa-T4+ cells with 5? splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3? base pairs of the exon (–3 to –1) and the eight most 5? base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5? splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, ?2-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.

Freund, Marcel; Asang, Corinna; Kammler, Susanne; Konermann, Carolin; Krummheuer, Jorg; Hipp, Marianne; Meyer, Imke; Gierling, Wolfram; Theiss, Stephan; Preuss, Thorsten; Schindler, Detlev; Kjems, J?rgen; Schaal, Heiner

2003-01-01

328

Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W  

PubMed Central

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

2009-01-01

329

The Intronic GABRG2 Mutation, IVS6+2T->G, Associated with CAE Altered Subunit mRNA Intron Splicing, Activated Nonsense-Mediated Decay and Produced a Stable Truncated ?2 Subunit  

PubMed Central

The intronic GABRG2 mutation, IVS6+2T?G, was identified in an Australian family with childhood absence epilepsy (CAE) and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wildtype and mutant ?2S subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wildtype and mutant ?2S subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wildtype and mutant GABRG2 mRNA splicing patterns were determined in both BAC transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention and produced a frame shift in exon 7 that created a premature translation-termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense mediated mRNA decay (NMD) or translated to a stable, truncated subunit (the ?2-PTC subunit) containing the first 6 GABRG2 exons and a novel frame-shifted 29 aa C terminal tail. The ?2-PTC subunit was homologous to the mollusk acetylcholine binding protein (AChBP) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with ?1 and ?2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T?G, reduced surface ???2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant negative effect on ???2 receptor assembly.

Tian, Mengnan; Macdonald, Robert L.

2012-01-01

330

Minimally invasive donor nephrectomy: innovations.  

PubMed

From open surgery to laparoscopic surgery, there has been an evolution in the surgical technique for live donor nephrectomy which goes beyond patient comfort. As a unique operation where the margin for error is nearly nil, and where the patient is essentially harmed for an altruistic goal, ensuring the best possible result is vital. Additionally, as the morbidity of the operation decreases, there is a theoretical increase in the donor pool. In this review, the latest techniques for minimally invasive live donor nephrectomy are covered, including new approaches such as laparoendoscopic single-site surgery, natural orifice surgery, and new tools such as robotics. PMID:24338815

Caso, Jorge R

2014-01-01

331

Cleavage of intron from the standard or non-standard position of the precursor tRNA by the splicing endonuclease of Aeropyrum pernix, a hyper-thermophilic Crenarchaeon, involves a novel RNA recognition site in the Crenarchaea specific loop.  

PubMed

In Crenarchaea, several tRNA genes are predicted to express precursor-tRNAs (pre-tRNAs) with canonical or non-canonical introns at various positions. We initially focused on the tRNA(Thr) species of hyperthermophilic crenarchaeon, Aeropyrum pernix (APE) and found that in the living APE cells three tRNA(Thr) species were transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them were cleaved from standard and non-standard positions. Biochemical studies revealed that the APE splicing endonuclease (APE-EndA) removed both types of introns, including the non-canonical introns, without any nucleotide modification. To clarify the underlying reasons for broad substrate specificity of APE-EndA, we determined the crystal structure of wild-type APE-EndA and subsequently compared its structure with that of Archaeaoglobus fulgidus (AFU)-EndA, which has narrow substrate specificity. Remarkably, structural comparison revealed that APE-EndA possesses a Crenarchaea specific loop (CSL). Introduction of CSL into AFU-EndA enhanced its intron-cleaving activity irrespective of the position or motif of the intron. Thus, our biochemical and crystallographic analyses of the chimera-EndA demonstrated that the CSL is responsible for the broad substrate specificity of APE-EndA. Furthermore, mutagenesis studies revealed that Lys44 in CSL functions as the RNA recognition site. PMID:21846775

Hirata, Akira; Kitajima, Tsubasa; Hori, Hiroyuki

2011-11-01

332

A splice site mutation in the methyltransferase gene FTSJ1 in Xp11.23 is associated with non-syndromic mental retardation in a large Belgian family (MRX9)  

PubMed Central

Mental retardation is the most frequent cause of serious handicap in children and young adults. The underlying causes of this heterogeneous condition are both acquired and genetically based. A recently performed refinement of the linkage interval in a large Belgian family with mild to severe non-syndromic X linked mental retardation, classified as MRX9, revealed a candidate region of 11.3 Mb between markers DXS228 and DXS1204 on the short arm of the X chromosome. In order to identify the underlying disease gene in the MRX9 family, we established a gene catalogue for the candidate region and performed comprehensive mutation analysis by direct sequencing. A human homologue of the bacterial 23S rRNA methyltransferase Fstj, the FTSJ1 gene, is located within this region and displayed a sequence alteration in the conserved acceptor splice site of intron 3 (IVS3-2A>G) in all tested patients and carrier females of this family. In contrast, it was absent in all unaffected male family members tested. The mutation results in skipping of exon 4 and introduces a premature stop codon in exon 5, probably leading to a severely truncated protein. Our finding indicates that a protein, possibly associated with ribosomal stability, can be linked to X linked mental retardation (XLMR).

Ramser, J; Winnepenninckx, B; Lenski, C; Errijgers, V; Platzer, M; Schwartz, C; Meindl, A; Kooy, R

2004-01-01

333

Extensive aggregation of ?-synuclein and tau in juvenile-onset neuroaxonal dystrophy: an autopsied individual with a novel mutation in the PLA2G6 gene-splicing site  

PubMed Central

Background Infantile neuroaxonal dystrophy (INAD) is a rare autosomal-recessive neurodegenerative disorder. Patients with INAD usually show neurological symptoms with infant onset and die in childhood. Recently, it was reported that mutations in the PLA2G6 gene cause INAD, but neuropathological analysis of genetically confirmed individuals with neuroaxonal dystrophy has been limited. Results Here, we report a Japanese individual with neuroaxonal dystrophy associated with compound heterozygous mutations in the PLA2G6 gene. A novel splice-site mutation resulting in skipping and missense mutations (p.R538C) in exon 9 was identified in the patient. This patient initially presented with cerebellar ataxia at the age of 3 years, which was followed by symptoms of mental retardation, extrapyramidal signs, and epileptic seizure. The patient survived until 20 years of age. Neuropathological findings were characterized by numerous axonal spheroids, brain iron deposition, cerebellar neuronal loss, phosphorylated alpha-synuclein-positive Lewy bodies (LBs), and phosphorylated-tau-positive neurofibrillary tangles. In particular, LB pathology exhibited a unique distribution with extremely severe cortical involvement. Conclusions Our results support a genetic clinical view that compound heterozygous mutations with potential residual protein function are associated with a relatively mild phenotype. Moreover, the severe LB pathology suggests that dysfunction of the PLA2G6 gene primarily contributes to LB formation.

2013-01-01

334

A novel de novo splice-site mutation in the COL7A1 gene in dominant dystrophic epidermolysis bullosa (DDEB): specific exon skipping could be a prognostic factor for DDEB pruriginosa.  

PubMed

We report a Japanese infant who had a novel de novo splice-site mutation in the COL7A1 gene, which resulted in in-frame exon 87 skipping. Very interestingly, most of the previously reported cases with the same exon skipping presented as dystrophic epidermolysis bullosa (DEB) pruriginosa. The proband in this study showed an extremely mild clinical phenotype, with no nail dystrophy, pruritus or prurigo-like lesions. However, dominant (DDEB) pruriginosa often shows a typical mild DEB phenotype until the onset of pruritus, making it likely that as she gets older the proband will present with features consistent with DDEB pruriginosa. By knowing in advance the anticipated clinical course, it might be possible to reduce or even prevent development of nodular prurigo-like lesions by sufficient control of pruritus. Our study should contribute to further refinement of the genotype-phenotype correlations in DEB, emphasizing the significance of mutation analysis for correct diagnosis and possibly for prediction of prognosis. PMID:19486058

Saito, M; Masunaga, T; Ishiko, A

2009-12-01

335

Impact of BRCA1 and BRCA2 variants on splicing: clues from an allelic imbalance study  

Microsoft Academic Search

Nearly one-half of BRCA1 and BRCA2 sequence variations are variants of uncertain significance (VUSs) and are candidates for splice alterations for example, by disrupting\\/creating splice sites. As out-of-frame splicing defects lead to a marked reduction of the level of the mutant mRNA cleared through nonsense-mediated mRNA decay, a cDNA-based test was developed to show the resulting allelic imbalance (AI). Fifty-four

Virginie Caux-Moncoutier; Sabine Pagès-Berhouet; Dorothée Michaux; Bernard Asselain; Laurent Castéra; Antoine De Pauw; Bruno Buecher; Marion Gauthier-Villars; Dominique Stoppa-Lyonnet; Claude Houdayer

2009-01-01

336

Human Na(+)-dependent vitamin C transporter 1 (hSVCT1): primary structure, functional characteristics and evidence for a non-functional splice variant.  

PubMed

We report here on the cloning and functional characterization of human Na(+)-dependent vitamin C transporter 1 (SVCT1). The human SVCT1 cDNA, obtained from a Caco2 cell cDNA library, encodes a protein of 598 amino acids with 12 putative transmembrane domains. The SVCT1-specific transcript, 2.4 kb in size, is expressed in kidney, liver, small intestine, thymus and prostate. When expressed heterologously in HRPE cells, SVCT1 mediates the transport of ascorbate, the reduced form of vitamin C, in a Na(+)-dependent manner. The transporter is specific for ascorbate with a K(t) of approximately 75 microM. The relationship between the cDNA-specific uptake rate of ascorbate and Na(+) concentration is sigmoidal with a Na(+):ascorbate stoichiometry of 2:1, indicating that the transport process is electrogenic. In Caco2 cells and in normal human intestine, SVCT1 also exists as a non-functional splice variant with a four amino acid sequence inserted between E-155 and V-156. The splice variant results from the use of a donor site 12 bp downstream of the normal donor site. PMID:10556483

Wang, H; Dutta, B; Huang, W; Devoe, L D; Leibach, F H; Ganapathy, V; Prasad, P D

1999-11-01

337

Alternatively spliced domains interact to regulate BK potassium channel gating.  

PubMed

Most human genes contain multiple alternative splice sites believed to extend the complexity and diversity of the proteome. However, little is known about how interactions among alternative exons regulate protein function. We used the Caenorhabditis elegans slo-1 large-conductance calcium and voltage-activated potassium (BK) channel gene, which contains three alternative splice sites (A, B, and C) and encodes at least 12 splice variants, to investigate the functional consequences of alternative splicing. These splice sites enable the insertion of exons encoding part of the regulator of K(+) conductance (RCK)1 Ca(2+) coordination domain (exons A1 and A2) and portions of the RCK1-RCK2 linker (exons B0, B1, B2, C0, and C1). Exons A1 and A2 are used in a mutually exclusive manner and are 67% identical. The other exons can extend the RCK1-RCK2 linker by up to 41 residues. Electrophysiological recordings of all isoforms show that the A1 and A2 exons regulate activation kinetics and Ca(2+) sensitivity, but only if alternate exons are inserted at site B or C. Thus, RCK1 interacts with the RCK1-RCK2 linker, and the effect of exon variation on gating depends on the combination of alternate exons present in each isoform. PMID:22049343

Johnson, Brandon E; Glauser, Dominique A; Dan-Glauser, Elise S; Halling, D Brent; Aldrich, Richard W; Goodman, Miriam B

2011-12-20

338

DNA Computing: Distributed Splicing Systems  

Microsoft Academic Search

Because splicing systems with a finite set of rules generate only regular languages, it is necessary to supplement such a system with a control mechanism on the use of rules. One fruitful idea is to use distributed architectures suggested by the grammar systems area. Three distributed computability (language generating) devices based on splicing are discussed here. First, we improve a

Gheorghe Paun

1997-01-01

339

Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution  

PubMed Central

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3? splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ?15-fold increase in the frequency of three base pair gaps at 3? splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.

Bradley, Robert K.; Merkin, Jason; Lambert, Nicole J.; Burge, Christopher B.

2012-01-01

340

Alternative splicing of rat tropoelastin mRNA is tissue-specific and developmentally regulated.  

PubMed

Sequence analysis of cDNA clones coding for rat tropoelastin previously has identified two variants that potentially corresponded to alternatively spliced tropoelastin mRNAs (Pierce et al., 1990). We have now used S1 nuclease protection analysis of total RNA from aorta, skin and lungs of 10-day and 6-week old rats to localize all sites of alternative splicing in the tropoelastin mRNA and to examine tissue-specific and developmental regulation of the use of these sites. This analysis revealed multiple sites of alternative splicing involving rat tropoelastin coding sequences corresponding to exons 12 through 15 of the bovine tropoelastin gene and a single site of alternative splicing at sequences corresponding to exon 33. Messenger RNAs from all three tissues at both developmental stages were alternatively spliced at the same sites; there was no evidence for the use of an alternative splice site unique to a particular tissue or developmental stage. However, both tissue-specific and developmentally regulated differences were apparent in the proportion of rat tropoelastin mRNA alternatively spliced at exon 33. Tropoelastin mRNA from the aorta and lungs of neonatal rats was alternatively spliced at exon 33 ten time more frequently than tropoelastin mRNA from skin. Between 10 days and 6 weeks of development, the use of this site of alternative splicing decreased by twenty-fold in RNA from skin, ten-fold in RNA from lungs and two-fold in RNA from aorta. In contrast, alternative splicing at exons 12 through 15 occurred in a small percentage of the mRNA and use of these sites exhibited minimal tissue-specific differences or developmental regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1811166

Heim, R A; Pierce, R A; Deak, S B; Riley, D J; Boyd, C D; Stolle, C A

1991-11-01

341

Basal Splicing Factors Regulate the Stability of Mature mRNAs in Trypanosomes*  

PubMed Central

Gene expression in trypanosomes is mainly regulated post-transcriptionally. Genes are transcribed as polycistronic mRNAs that are dissected by the concerted action of trans-splicing and polyadenylation. In trans-splicing, a common exon, the spliced leader, is added to all mRNAs from a small RNA. In this study, we examined by microarray analysis the transcriptome following RNAi silencing of the basal splicing factors U2AF65, SF1, and U2AF35. The transcriptome data revealed correlations between the affected genes and their splicing and polyadenylation signaling properties, suggesting that differential binding of these factors to pre-mRNA regulates trans-splicing and hence expression of specific genes. Surprisingly, all these factors were shown to affect not only splicing but also mRNA stability. Affinity purification of SF1 and U2AF35 complexes supported their role in mRNA stability. U2AF35 but not SF1 was shown to bind to ribosomes. To examine the role of splicing factors in mRNA stability, mutations were introduced into the polypyrimidine tract located in the 3? UTR of a mini-gene, and the results demonstrate that U2AF65 binds to such a site and controls the mRNA stability. We propose that transcripts carrying splicing signals in their 3? UTR bind the splicing factors and control their stability.

Gupta, Sachin Kumar; Carmi, Shai; Ben-Asher, Hiba Waldman; Tkacz, Itai Dov; Naboishchikov, Ilana; Michaeli, Shulamit

2013-01-01

342

Basal splicing factors regulate the stability of mature mRNAs in trypanosomes.  

PubMed

Gene expression in trypanosomes is mainly regulated post-transcriptionally. Genes are transcribed as polycistronic mRNAs that are dissected by the concerted action of trans-splicing and polyadenylation. In trans-splicing, a common exon, the spliced leader, is added to all mRNAs from a small RNA. In this study, we examined by microarray analysis the transcriptome following RNAi silencing of the basal splicing factors U2AF65, SF1, and U2AF35. The transcriptome data revealed correlations between the affected genes and their splicing and polyadenylation signaling properties, suggesting that differential binding of these factors to pre-mRNA regulates trans-splicing and hence expression of specific genes. Surprisingly, all these factors were shown to affect not only splicing but also mRNA stability. Affinity purification of SF1 and U2AF35 complexes supported their role in mRNA stability. U2AF35 but not SF1 was shown to bind to ribosomes. To examine the role of splicing factors in mRNA stability, mutations were introduced into the polypyrimidine tract located in the 3' UTR of a mini-gene, and the results demonstrate that U2AF65 binds to such a site and controls the mRNA stability. We propose that transcripts carrying splicing signals in their 3' UTR bind the splicing factors and control their stability. PMID:23283975

Gupta, Sachin Kumar; Carmi, Shai; Waldman Ben-Asher, Hiba; Tkacz, Itai Dov; Naboishchikov, Ilana; Michaeli, Shulamit

2013-02-15

343

Global analysis of alternative splicing regulation by insulin and wingless signaling in Drosophila cells  

PubMed Central

Background Despite the prevalence and biological relevance of both signaling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signaling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis using splicing-sensitive microarrays of changes in alternative splicing induced by activation of two distinct signaling pathways, insulin and wingless, in Drosophila cells in culture. Results Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and post-transcriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related to carbohydrate metabolism and cellular signaling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5' splice sites. Conclusions Taken together, our data indicate that signaling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for crosstalk between different signaling pathways.

Hartmann, Britta; Castelo, Robert; Blanchette, Marco; Boue, Stephanie; Rio, Donald C; Valcarcel, Juan

2009-01-01

344

Systematical identification of splicing regulatory cis-elements and cognate trans-factors.  

PubMed

The majority of human genes undergo alternative splicing to generate multiple isoforms with distinct functions. This process is generally controlled by cis-acting splicing regulatory elements (SREs) that recruit trans-acting factors to promote or inhibit the use of nearby splice sites. The growing interest in understanding the regulatory rules of splicing necessitates the systematic identification of these SREs and their cognate protein factors using experimental and computational approaches. Here we describe a strategy to identify and analyze both cis-acting SREs and trans-acting splicing factors. This strategy involves a cell-based screen to identify SREs from a random sequences library and a modified RNA affinity purification approach to unbiasedly identify the splicing factors. These methods can be adopted to identify splicing enhancers or silencers in both exons and introns, and can be extended to different cultured cells. The resulting SREs and splicing factors can be further analyzed with a series of computational and experimental approaches. This approach will help us to collect a molecular part-list for splicing regulation, providing a rich data source that enables a better understanding of the "splicing code". PMID:23974071

Wang, Yang; Wang, Zefeng

2014-02-01

345

Alternative splicing and muscular dystrophy  

PubMed Central

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.

Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

2013-01-01

346

Committee of Donor Agencies for Small Enterprise Development  

NSDL National Science Digital Library

Established in 1979, the Committee of Donor Agencies promotes the development of small enterprise in developing countries. This site offers numerous working and research papers about the Committee of Donor Agencies and its members. Showcased on the site are the Committee's Donor Business Development Services Case Studies. The Case studies are browseable by several categories including Region, Country, Theme, and Member Agency. Also provided are the Donor Committee Guidelines and links to member agencies's sites.

347

Ecdysteroid-responsive genes, RXR and E75, in the tropical land crab, Gecarcinus lateralis: differential tissue expression of multiple RXR isoforms generated at three alternative splicing sites in the hinge and ligand-binding domains.  

PubMed

In order to study the potential role of the steroid molting hormone (20-hydroxyecdysone) in regulating molt-induced claw muscle atrophy, full-length cDNAs encoding retinoid-X receptor (Gl-RXR) and E75 early ecdysone inducible gene (Gl-E75) were obtained from land crab (Gecarcinus lateralis) skeletal muscle mRNA using RT-PCR and 3' and 5' RACE. Gl-E75A (3528bp), which encoded a protein of 828 amino acids, had highest sequence identity to Me-E75A from a shrimp (Metapenaeus ensis). It was expressed in skeletal muscle and gonads. The deduced amino acid sequence of Gl-RXR was highly similar to that of the fiddler crab RXR (Up-RXR) and insect ultraspiracle (USP). Nine variant sequences occurred in Gl-RXR mRNAs at three alternative splicing sites, one in the "T box" in the linker D domain and two in the ligand-binding domain (LBD). The three T-box variants, termed T(+8), T(+7), and T(+12), contained insertions of 8, 7, or 12 amino acids, respectively. Four variants were generated at the first site in the LBD. Two of the LBD site 1 variants differed in the presence (+33) or absence (-33) of a 33-amino acid sequence; the other two were LBD truncations with or without the 33 amino acid sequence (+33DeltaE/F and -33DeltaE/F, respectively). Two variants differing in the presence (+35) or absence (-35) of a 35-amino acid sequence were generated at the second site in the LBD. The Gl-RXRa isoform (1516 bp) with the longest open reading frame (+12/+33/+35) encoded a protein of 436 amino acids. Thoracic muscle expressed only isoforms with the T(+12) sequence. In contrast, claw muscle expressed isoforms with T(+7) or T(+12) and fewer isoforms with T(+8). Ovary and testis expressed a greater number of RXR isoforms than skeletal muscle. All tissues expressed full-length and truncated RXR isoforms. These data suggest that differences in response of claw and thoracic muscles to elevated ecdysteroid are due in part to differences in the expression of RXR isoforms. PMID:16150535

Kim, Hyun-Woo; Lee, Sung Gu; Mykles, Donald L

2005-10-20

348

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

PubMed

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

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

2014-01-01

349

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

PubMed Central

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

Kamtchueng, Catherine; Stebenne, Marie-Eve; Delannoy, Aurelie; Wilhelm, Emmanuelle; Leger, Helene; Benecke, Arndt G.; Bell, Brendan

2014-01-01

350

Simultaneous detection of Hb constant spring (?142, TAA>CAA, ?2) and the ?2 IVS-I donor site (-TGAGG) deletion by a simple polymerase chain reaction-based method in Iran.  

PubMed

Hb Constant Spring (Hb CS, codon 142, TAA>CAA, ?2) (HBA2:c.427T>C) and ?2 IVS-I donor site (GAGGTGAGG>GAGG?- - - - -) (HBA2:c.95+2_95+6delTGAGG) are nondeletional ?-thalassemia (?-thal) mutations found all over the world. Identification of ?-thal genotypes in at-risk couples for severe anemia or in highly heterogeneous populations requires rapid, accurate and cost-effective genotyping methods. In this study, a pair of primers were used to specifically amplify an 883 bp fragment from the ?2-globin gene in order to simultaneously identify these two mutations by a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. We determined the genotypic frequencies of Hb CS and the ?2 IVS-I donor site mutations after amplification and enzymatic digestion with Tru9I in 238 northern Iranian samples referred for ?-thal testing. Hb CS and the ?2 IVS-I donor site mutations accounted for 21 (8.8%) and 29 (12.2%) of the nondeletional cases. This genotyping assay has proven to be a rapid, reliable and useful diagnostic tool for simultaneous detection of these two anomalies for genetic counseling or further prenatal diagnosis. PMID:22356652

Akhavan-Niaki, Haleh; Banihashemi, Ali; Mostafazadeh, Amrollah; Kholghi Oskooei, Vahid; Azizi, Mandana; Youssefi Kamangar, Reza; Elmi, Maryam Mitra

2012-01-01

351

The splice leader addition domain represents an essential conserved motif for heterologous gene expression in B. malayi  

PubMed Central

Two promoters from the human filarial parasite Brugia malayi have been mapped in detail. The essential domains of both promoters lacked canonical eukaryotic core promoter motifs. However, the largest contiguous essential domain in both promoters flanked and included the splice leader addition site. These findings suggested that the region flanking the trans-splicing addition site might represent a conserved core domain in B. malayi promoters. To test this hypothesis, the putative promoters of 12 trans-spliced genes encoding ribosomal protein homologues from B. malayi were isolated and tested for activity in a B. malayi transient transfection system. Of the 12 domains examined, 11 produced detectable reporter gene activity. Mutant constructs of the six most active promoters were prepared in which the spliced leader acceptor site and the10 nt upstream and downstream of the site were deleted. All deletion constructs exhibited >90% reduction in reporter gene activity relative to their respective wild type sequences. A conserved pyrimidine-rich tract was located directly upstream from the spliced leader splice acceptor site which contained a conserved T residue located at position ?3. Mutation of the entire polypyrimidine tract or the conserved T individually resulted in the loss of over 90% of reporter gene activity. In contrast, mutation of the splice acceptor site did not significantly reduce promoter activity. These data suggest that the region surrounding the splice acceptor site in the ribosomal promoters represents a conserved essential domain which functions independently of splice leader addition.

Liu, Canhui; Chauhan, Chitra; Katholi, Charles R.; Unnasch, Thomas R.

2009-01-01

352

Classifying MLH1 and MSH2 variants using bioinformatic prediction, splicing assays, segregation and tumor characteristics  

PubMed Central

Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for ten variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16/19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach which includes in vitro, tumor pathology, clinical, and evolutionary conservation data.

Arnold, Sven; Buchanan, Daniel D.; Barker, Melissa; Jaskowski, Lesley; Walsh, Michael D.; Birney, Genevieve; Woods, Michael O.; Hopper, John L.; Jenkins, Mark A.; Brown, Melissa A.; Tavtigian, Sean V.; Goldgar, David E.; Young, Joanne P.; Spurdle, Amanda B.

2009-01-01

353

Finding a Donor  

MedlinePLUS

... and foundation partners Global transplant network Donor centers Recruitment centers International donor centers Cord blood banks Cooperative ... information Annual report Funding patient assistance Funding donor recruitment Careers Working with us Our accomplishments & recognition Career ...

354

Contacting My Donor Family  

MedlinePLUS

... My Donor Family Newsroom Minorities Contacting My Donor Family Writing anything can be a challenge. Staring at ... down to write a note to your donor family can feeling overwhelming. The good news is that ...

355

Caffeine induces tumor cytotoxicity via the regulation of alternative splicing in subsets of cancer-associated genes.  

PubMed

Caffeine causes a diverse range of pharmacological effects that are time- and concentration-dependent and reversible. The detailed mechanisms of caffeine in tumor suppression via tumor suppressor protein p53 remain unclear. The isoforms of p53 are physiological proteins that are expressed in normal cells and generated via alternative promoters, splicing sites and/or translational initiation sites. In this study, we investigated how caffeine modulated cell cycle arrest and apoptosis via the expression of various alternatively spliced p53 isoforms. Caffeine reduced p53? expression and induced the expression of p53?, which contains an alternatively spliced p53 C-terminus. In HeLa cells, the expression levels of many serine/arginine-rich splicing factors, including serine/arginine-rich splicing factors 2 and 3, were altered by caffeine. Serine/arginine-rich splicing factor 3 was a promising candidate for the serine/arginine-rich splicing factors responsible for the alternative splicing of p53 in response to caffeine treatment. In addition to p53-dependent functions, multiple target genes of serine/arginine-rich splicing factor 3 suggest that caffeine can regulate epithelial-mesenchymal-transition and hypoxic conditions to inhibit the survival of tumor cells. In summary, our data provide a new pathway of caffeine-modulated tumor suppression via the alternative splicing of the target genes of serine/arginine-rich splicing factor 3. PMID:24333670

Lu, Guan-Yu; Huang, Shih-Ming; Liu, Shu-Ting; Liu, Pei-Yao; Chou, Wei-Yuan; Lin, Wei-Shiang

2014-02-01

356

Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism  

USGS Publications Warehouse

The gene encoding IgH ? 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 ?-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 ? 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 ? transcript resulted in two ?-H chains, which incorporated C?1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory ? 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.

Ramirez-Gomez, F.; Greene, W.; Rego, K.; Hansen, J. D.; Costa, G.; Kataria, P.; Bromage, E. S.

2012-01-01

357

Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo  

PubMed Central

The heterodimeric splicing factor U2AF plays an important role in 3? splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3? splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3? splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3? pyrimidine tract. These and other studies performed in fission yeast support a model for 3? splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.

Webb, Christopher J.; Lakhe-Reddy, Sujata; Romfo, Charles M.; Wise, Jo Ann

2005-01-01

358

A new multimode optical fiber splicing technique  

NASA Astrophysics Data System (ADS)

For the French local cable network, it is necessary to perform a great number of multimode optical fiber splices. We present a low cost splicing technique which has been industrialized to satisfy the requirements of the videocommunication networks. In this splice, fibers are aligned in an elastomeric groove and then, bonded to a glass plate. Training time is very short and field installation is easy. It is a good quality, reliable splice and more than 100,000 splices has been already realized in the field. In this paper we present this splicing technique and the performances of the splice.

Ruello, Y.; Malavieille, F. L.

1986-11-01

359

Tissue-Specific Genetic Control of Splicing: Implications for the Study of Complex Traits  

PubMed Central

Numerous genome-wide screens for polymorphisms that influence gene expression have provided key insights into the genetic control of transcription. Despite this work, the relevance of specific polymorphisms to in vivo expression and splicing remains unclear. We carried out the first genome-wide screen, to our knowledge, for SNPs that associate with alternative splicing and gene expression in human primary cells, evaluating 93 autopsy-collected cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononucleated cell samples collected from living healthy donors. We identified 23 high confidence associations with total expression and 80 with alternative splicing as reflected by expression levels of specific exons. Fewer than 50% of the implicated SNPs however show effects in both tissue types, reflecting strong evidence for distinct genetic control of splicing and expression in the two tissue types. The data generated here also suggest the possibility that splicing effects may be responsible for up to 13 out of 84 reported genome-wide significant associations with human traits. These results emphasize the importance of establishing a database of polymorphisms affecting splicing and expression in primary tissue types and suggest that splicing effects may be of more phenotypic significance than overall gene expression changes.

Cronin, Kenneth D; Maia, Jessica M; Shianna, Kevin V; Gabriel, Willow N; Welsh-Bohmer, Kathleen A; Hulette, Christine M; Denny, Thomas N; Goldstein, David B

2008-01-01

360

Systematic screening of FBN1 gene unclassified missense variants for splice abnormalities.  

PubMed

Defects at the level of pre-mRNA splicing are a common source of genetic mutation but such mutations are not always easy to identify from DNA sequence data alone. Clinical practice has only recently begun to incorporate analysis for this type of abnormality. Some base changes at the DNA level currently viewed as unclassified variants or missense mutations may influence RNA splicing. To address this problem for fibrillin 1 (FBN1) gene missense mutations we have carried out RNA analysis and in silico analysis with splice site prediction programs on 40 cases with 36 different mutations. Direct analysis of RNA from blood was performed by cDNA preparation, PCR amplification of specific FBN1 fragments, gel electrophoresis and sequencing of the PCR products. Of the 36 missense base changes, direct RNA analysis identified 2 which caused an abnormality of splicing. In silico analysis using five splice site prediction programs did not always accurately predict the splicing seen by direct RNA analysis. In conclusion, some apparent missense mutations have an effect on splicing which can be identified by direct RNA analysis, however, in silico analysis of splice sites is not always accurate, should be carried out with more than one prediction program and results should be used with caution. PMID:21895641

Robinson, D O; Lin, F; Lyon, M; Raponi, M; Cross, E; White, H E; Cox, H; Clayton-Smith, J; Baralle, D

2012-09-01

361

In vitro splicing of simian virus 40 early pre mRNA.  

PubMed Central

The products of splicing of simian virus 40 early pre mRNA in HeLa cell nuclear extracts have been characterized. Of the two alternative splicing patterns exhibited by this precursor in vivo, which involve the use of alternative large T and small t 5' splice sites and a single shared 3' splice site, only one, producing large T mRNA, was found to occur in vitro. A number of possible intermediates and byproducts of splicing of large T mRNA were observed, including free large T 5' exon, lariat form intron joined to 3' exon and free lariat and linear forms of large T intron. The formation of these products argues strongly for a basic similarity in the mechanism underlying large T and other, non-alternative splices. A collection of RNAs resulting from protection of early pre mRNA at specific points from an endogenous 5' to 3' exonuclease activity in vitro have also been observed. The regions of the precursor RNA protected map to positions immediately upstream of the 5' splice sites of large T and small t and the lariat branchpoint, and may represent interaction of these regions with components of the splicing machinery. Images

Noble, J C; Prives, C; Manley, J L

1986-01-01

362

Detection and quantification of alternative splice sites in Arabidopsis genes AtDCL2 and AtPTB2 with highly sensitive surface enhanced Raman spectroscopy (SERS) and gold nanoprobes.  

PubMed

Alternative splicing (AS) increases the size of the transcriptome and proteome to enhance the physiological capacity of cells. We demonstrate surface enhanced Raman spectroscopy (SERS) in combination with a DNA hybridization analytical platform to identify and quantify AS genes in plants. AS in AtDCL2 and AtPTB2 were investigated using non-fluorescent Raman probes using a 'sandwich assay'. Utilizing Raman probes conjugated to gold nanoparticles we demonstrate the recognition of RNA sequences specific to AtDCL2 and AtPTB2 splice junction variants with detection sensitivity of up to 0.1fM. PMID:24631541

Kadam, Ulhas S; Schulz, Burkhard; Lrudayaraj, Joseph

2014-05-01

363

Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis  

PubMed Central

Background The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. Results We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5? splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Conclusion Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.

2014-01-01

364

Splicing Efficiently Couples Optical Fibers  

NASA Technical Reports Server (NTRS)

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.

Lutes, G. F.

1985-01-01

365

Functional analysis and in vitro correction of splicing FAH mutations causing tyrosinemia type I.  

PubMed

Hereditary tyrosinemia type I (HT1) is a rare disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH) in the tyrosine catabolic pathway, resulting mainly in hepatic alterations due to accumulation of the toxic metabolites fumarylacetoacetate, maleylacetoacetate and succinylacetone.