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Sample records for exonic region 15-16

  1. Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15-16

    SciTech Connect

    Wijnen, J.; Khan, P.M.; Klift, H. van der

    1996-02-01

    Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer susceptibility condition. Inherited mutations in at least four DNA mismatch repair genes, hMSH2, hMLH1, hPMS1, and hPMS2, are known to cause HNPCC. In this study we used denaturing gradient gel electrophoresis (DGGE) to screen for hMLH1 mutations in 34 unrelated HNPCC families (30 Dutch, 3 Italian, and 1 Danish). Ten novel pathogenic germ-line mutations (seven affecting splice sites, two frameshifts, and one in-frame deletion of a single amino acid) have been identified in 12 (35%) of these families. In a previous study, hMSH2 mutations were found in 21% of the same families. While the spectrum of mutations at the hMSH2 gene among HNPCC patients appears heterogeneous, a cluster of hMLH1 mutations has been found in the region encompassing exons 15 and 16, which accounts for 50% of all the independent hMLH1 mutations described to date and for >20% of the unrelated HNPCC kindreds here analyzed. This unexpected finding has a great practical value in the clinical scenario of genetic services. 34 refs., 3 figs., 2 tabs.

  2. Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15-16.

    PubMed Central

    Wijnen, J.; Khan, P. M.; Vasen, H.; Menko, F.; van der Klift, H.; van den Broek, M.; van Leeuwen-Cornelisse, I.; Nagengast, F.; Meijers-Heijboer, E. J.; Lindhout, D.; Griffioen, G.; Cats, A.; Kleibeuker, J.; Varesco, L.; Bertario, L.; Bisgaard, M. L.; Mohr, J.; Kolodner, R.; Fodde, R.

    1996-01-01

    Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer susceptibility condition. Inherited mutations in at least four DNA mismatch repair genes, hMSH2, hMLH1, hPMS1, and hPMS2, are known to cause HNPCC. In this study we used denaturing gradient gel electrophoresis (DGGE) to screen for hMLH1 mutations in 34 unrelated HNPCC families (30 Dutch, 3 Italian, and 1 Danish). Ten novel pathogenic germ-line mutations (seven affecting splice sites, two frameshifts, and one in-frame deletion of a single amino acid) have been identified in 12 (35%) of these families. In a previous study, hMSH2 mutations were found in 21% of the same families. While the spectrum of mutations at the hMSH2 gene among HNPCC patients appears heterogeneous, a cluster of hMLH1 mutations has been found in the region encompassing exons 15 and 16, which accounts for 50% of all the independent hMLH1 mutations described to date and for > 20% of the unrelated HNPCC kindreds here analyzed. This unexpected finding has a great practical value in the clinical scenario of genetic services. Images Figure 1 Figure 3 PMID:8571956

  3. Distribution bias of the sequence matching between exons and introns in exon joint and EJC binding region in C. elegans.

    PubMed

    Zhang, Qiang; Li, Hong; Zhao, Xiaoqing; Zheng, Yan; Zhou, Deliang

    2015-01-01

    We propose a mechanism that there are matching relations between mRNA sequences and corresponding post-spliced introns, and introns play a significant role in the process of gene expression. In order to reveal the sequence matching features, Smith-Waterman local alignment method is used on C. elegans mRNA sequences to obtain optimal matched segments between exon-exon sequences and their corresponding introns. Distribution characters of matching frequency on exon-exon sequences and sequence characters of optimal matched segments are studied. Results show that distributions of matching frequency on exon-exon junction region have obvious differences, and the exon boundary is revealed. Distributions of the length and matching rate of optimal matched segments are consistent with sequence features of siRNA and miRNA. The optimal matched segments have special sequence characters compared with their host sequences. As for the first introns and long introns, matching frequency values of optimal matched segments with high GC content, rich CG dinucleotides and high λCG values show the minimum distribution in exon junction complex (EJC) binding region. High λCG values in optimal matched segments are main characters in distinguishing EJC binding region. Results indicate that EJC and introns have competitive and cooperative relations in the process of combining on protein coding sequences. Also intron sequences and protein coding sequences do have concerted evolution relations.

  4. Isolation of genes from the Batten candidate region using exon amplification

    SciTech Connect

    Lerner, T.J.; D`Arigo, K.L.; Haines, J.L.

    1995-06-05

    In order to identify genes originating from the Batten disease candidate region, we have used the technique of exon amplification to identify transcribed sequences. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. The source of DNA for these experiments was a collection of chromosome 16 cosmid contigs isolated by the direct subcloning of region-specific yeast artificial chromosomes (YACs) and hybridization of inter-alu PCR products from these YACs to the flow-sorted Los Alamos chromosome 16 cosmid library. We are now using the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes. 23 refs., 1 fig.

  5. The identification of exons from the MED/PSACH region of human chromosome 19

    SciTech Connect

    Li, Quan-Yi; Brook, J.D.; Lennon, G.G.

    1996-03-01

    We have used exon amplification to identify putative transcribed sequences from an 823-kb contig consisting of 28 cosmids that form a minimum tiling path from the interval 19p12-p13.1. This region contains the genes responsible for multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). We have trapped 66 exons (an average of 2.4 exons per cosmid) from pools of 2 or 3 cosmids. The majority of exons (51.5%) show only weak similarity or no similarity (36.3%) to sequences in current databases. Six of 8 exons examined from these groups, however, show cross-species sequence conservation, indicating that many of them probably represent authentic exons. Eight exons show identity or significant similarity to ESTs or known genes, including the human TNF receptor 3{prime}-flanking region gene, human epoxide hydrolase (EPHX), human growth/differentiation factor (GOF-1), human myocyte-specific enhancer factor 2, the rat neurocan gene, and the human cartilage oligomeric matrix protein gene (COMP). Mutations in this latter gene have recently been shown to be responsible for MED and PSACH. 33 refs., 4 figs., 2 tabs.

  6. Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion.

    PubMed

    Camp, Shelley; Zhang, Limin; Marquez, Michael; de la Torre, Brian; Long, Jeffery M; Bucht, Goran; Taylor, Palmer

    2005-12-15

    AChE is an alternatively spliced gene. Exons 2, 3 and 4 are invariantly spliced, and this sequence is responsible for catalytic function. The 3' alternatively spliced exons, 5 and 6, are responsible for AChE disposition in tissue [J. Massoulie, The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11 (3) (2002) 130-143; Y. Li, S. Camp, P. Taylor, Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene. J. Biol. Chem. 268 (8) (1993) 5790-5797]. The splice to exon 5 produces the GPI anchored form of AChE found in the hematopoietic system, whereas the splice to exon 6 produces a sequence that binds to the structural subunits PRiMA and ColQ, producing AChE expression in brain and muscle. A third alternative RNA species is present that is not spliced at the 3' end; the intron 3' of exon 4 is used as coding sequence and produces the read-through, unanchored form of AChE. In order to further understand the role of alternative splicing in the expression of the AChE gene, we have used homologous recombination in stem cells to produce gene specific deletions in mice. Alternatively and together exon 5 and exon 6 were deleted. A cassette containing the neomycin gene flanked by loxP sites was used to replace the exon(s) of interest. Tissue analysis of mice with exon 5 deleted and the neomycin cassette retained showed very low levels of AChE expression, far less than would have been anticipated. Only the read-through species of the enzyme was produced; clearly the inclusion of the selection cassette disrupted splicing of exon 4 to exon 6. The selection cassette was then deleted in exon 5, exon 6 and exons 5 + 6 deleted mice by breeding to Ella-cre transgenic mice. AChE expression in serum, brain and muscle has been analyzed. Another AChE gene targeted mouse strain involving a region in the first intron, found to be critical for AChE expression in muscle cells [S. Camp, L. Zhang, M. Marquez, B

  7. Single nucleotide polymorphism mining and nucleotide sequence analysis of Mx1 gene in exonic regions of Japanese quail

    PubMed Central

    Niraj, Diwesh Kumar; Kumar, Pushpendra; Mishra, Chinmoy; Narayan, Raj; Bhattacharya, Tarun Kumar; Shrivastava, Kush; Bhushan, Bharat; Tiwari, Ashok Kumar; Saxena, Vishesh; Sahoo, Nihar Ranjan; Sharma, Deepak

    2015-01-01

    Aim: An attempt has been made to study the Myxovirus resistant (Mx1) gene polymorphism in Japanese quail. Materials and Methods: In the present, investigation four fragments viz. Fragment I of 185 bp (Exon 3 region), Fragment II of 148 bp (Exon 5 region), Fragment III of 161 bp (Exon 7 region), and Fragment IV of 176 bp (Exon 13 region) of Mx1 gene were amplified and screened for polymorphism by polymerase chain reaction-single-strand conformation polymorphism technique in 170 Japanese quail birds. Results: Out of the four fragments, one fragment (Fragment II) was found to be polymorphic. Remaining three fragments (Fragment I, III, and IV) were found to be monomorphic which was confirmed by custom sequencing. Overall nucleotide sequence analysis of Mx1 gene of Japanese quail showed 100% homology with common quail and more than 80% homology with reported sequence of chicken breeds. Conclusion: The Mx1 gene is mostly conserved in Japanese quail. There is an urgent need of comprehensive analysis of other regions of Mx1 gene along with its possible association with the traits of economic importance in Japanese quail. PMID:27047057

  8. Exon organization of the mouse entactin gene corresponds to the structural domains of the polypeptide and has regional homology to the low-density lipoprotein receptor gene

    SciTech Connect

    Durkin, M.E.; Chung, A.E.; Wewer, U.M.

    1995-03-20

    Entactin is a widespread basement membrane protein of 150 kDa that binds to type IV collagen and laminin. The complete exon-intron structure of the mouse entactin gene has been determined from {lambda} genomic DNA clones. The gene spans at least 65 kb and contains 20 exons. The exon organization of the mouse entactin gene closely corresponds to the organization of the polypeptide into distinct structural and functional domains. The two amino-terminal globular domains are encoded by three exons each. Single exons encode the two protease-sensitive, O-glycosylated linking regions. The six EGF-like repeats and the single thyroglobulin-type repeat are each encoded by separate exons. The carboxyl-terminal half of entactin displays sequence homology to the growth factor-like region of the low-density lipoprotein receptor, and in both genes this region is encoded by eight exons. The positions of four introns are also conserved in the homologous region of the two genes. These observations suggest that the entactin gene has evolved via exon shuffling. Finally, several sequence polymorphisms useful for gene linkage analysis were found in the 3{prime} noncoding region of the last exon. 52 refs., 8 figs.

  9. The human decorin gene: Intron-exon organization, discovery of two alternatively spliced exons in the 5[prime] untralsated region, and mapping of the gene to chromosome 12q23

    SciTech Connect

    Danielson, K.G.; Fazzio, A.; Cohen, I.; Cannizzaro, L.A.; Eichstetter, I.; Iozzo, R.V. )

    1993-01-01

    Decorin is a chondroitin/dermatan sulfate proteoglycan expressed by most vascular and avascular connective tissues and, because of its ability to interact with collagen and growth factors, has been implicated in the control of matrix assembly and cellular growth. To understand the molecular mechanisms involved in regulating its tissue expression, we have isolated a number of genomic clones encoding the complete decorin gene. The human decorin gene spans over 38 kb of continuous DNA sequence and contains eight exons and very large introns, two of which are 5.4 and > 13.2 kb. We have discovered two alternatively spliced leader exons, exons Ia and Ib, in the 5[prime] untranslated region. These exons were identified by cloning and sequencing cDNAs obtained by polymerase chain reaction amplification of a fibroblast cDNA library. Using Northern blotting or reverse transcriptase PCR, we detected the two leader exons in a variety of mRNAs isolated from human cell lines and tissues. Interestingly, sequences highly (74-87%) homologous to exons Ia and lb are found in the 5[prime]untranslated region of avian and bovine decorin, respectively. This high degree of conservation among species suggests regulatory functions for these leader exons. In the 3' untranslated region there are several polyadenylation sites, and at least two of these sites could give rise to the transcripts of [approx]1.6 and [approx]1.9 kb, typically detected in a variety of tissues and cells. Using a genomic clone as the labeled probe and in situ hybridization of human metaphase chromosomes, we have mapped the decorin gene to the discrete region of human chromosome 12q23. This sturdy provides the molecular basis for discerning the transcriptional control of the decorin gene and offers the opportunity to investigate genetic disorders linked to this important human gene. 57 refs., 11 figs., 3 tabs.

  10. Cloning of the alpha-adducin gene from the Huntington's disease candidate region of chromosome 4 by exon amplification.

    PubMed

    Taylor, S A; Snell, R G; Buckler, A; Ambrose, C; Duyao, M; Church, D; Lin, C S; Altherr, M; Bates, G P; Groot, N

    1992-11-01

    We have applied the technique of exon amplification to the isolation of genes from the chromosome 4p16.3 Huntington's disease (HD) candidate region. Exons recovered from cosmid Y24 identified cDNA clones corresponding to the alpha-subunit of adducin, a calmodulin-binding protein that is thought to promote assembly of spectrin-actin complexes in the formation of the membrane cytoskeleton, alpha-adducin is widely expressed and, at least in brain, is encoded by alternatively spliced mRNAs. The alpha-adducin gene maps immediately telomeric to D4S95, in a region likely to contain the HD defect, and must be scrutinized to establish whether it is the site of the HD mutation.

  11. Abnormal Methylation Status of the GNAS Exon 1A Region in Pseudohypohyperparathyroidism Combined With Turner Syndrome.

    PubMed

    Zhu, Jie; Wang, Dong; Ren, An; Xing, Yan; Zhang, Dongliang; Wei, Jun; Yu, Ning; Xing, Xuenong; Ye, Shandong

    2015-12-01

    Pseudohypohyperparathyroidism (PHHP) is a rare type of pseudohypoparathyroidism (PHP), which seems to have a normal skeletal response to parathyroid hormone but shows renal resistance. Almost all patients with PHHP have PHP Ib, a subtype of PHP that is usually caused by GNAS methylation defects, often in exon 1A. Some features of Albright hereditary osteodystrophy can occasionally be found in patients with PHHP, but these features are also common in Turner syndrome. The authors report on an extremely rare case of a patient with PHHP and Turner syndrome, a 47-year-old woman who sought medical attention for hypocalcemia and elevated parathyroid hormone. She had no family history of hypocalcemia and no STX16 gene deletions. She had a mosaic karyotype of 46, X, del(X)(p11.4)/45, XO. Pyrosequencing was performed to determine the GNAS exon 1A methylation. The degree of methylation found in exon 1A of the patient was lower than her unaffected relatives.

  12. Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3

    PubMed Central

    Seabury, Christopher M.; Honeycutt, Rodney L.; Rooney, Alejandro P.; Halbert, Natalie D.; Derr, James N.

    2004-01-01

    Amino acid replacements encoded by the prion protein gene (PRNP) have been associated with transmissible and hereditary spongiform encephalopathies in mammalian species. However, an association between bovine spongiform encephalopathy (BSE) and bovine PRNP exon 3 has not been detected. Moreover, little is currently known regarding the mechanisms of evolution influencing the bovine PRNP gene. Therefore, in this study we evaluated the patterns of nucleotide variation associated with PRNP exon 3 for 36 breeds of domestic cattle and representative samples for 10 additional species of Bovinae. The results of our study indicate that strong purifying selection has intensely constrained PRNP over the long-term evolutionary history of the subfamily Bovinae, especially in regions considered to be of functional, structural, and pathogenic importance in humans as well as other mammals. The driving force behind this intense level of purifying selection remains to be explained. PMID:15477588

  13. DNA Methylation in the Exon 1 Region and Complex Regulation of Twist1 Expression in Gastric Cancer Cells

    PubMed Central

    Sakamoto, Ayuna; Akiyama, Yoshimitsu; Shimada, Shu; Zhu, Wei-Guo; Yuasa, Yasuhito; Tanaka, Shinji

    2015-01-01

    Twist1 overexpression is frequently observed in various cancers including gastric cancer (GC). Although DNA methylation of the Twist1 gene has been reported in cancer cells, the mechanisms underlying transcriptional activation remain uncertain. In this study, we first examined epigenetic alterations of the Twist1 using Twist1 transcription-positive and -negative cell lines that are derived from our established diffuse-type GC mouse model. Treatment with a DNA demethylation agent 5-aza-dC re-activated Twist1 expression in Twist1 expression-negative GC cells. According to methylation-specific PCR and bisulfite sequencing analysis, methylation at the CpG-rich region within Twist1 coding exon 1, rather than its promoter region, was tightly linked to transcriptional silencing of the Twist1 expression in mouse GC cells. Chromatin immunoprecipitation assays revealed that active histone mark H3K4me3 was enriched in Twist1 expression-positive cells, and inactive histone mark H3K9me3 was enriched in Twist1 expression-negative cells. The expression levels of Suv39h1 and Suv39h2, histone methyltransferases for H3K9me3, were inversely correlated with Twist1 expression, and knockdown of Suv39h1 or Suv39h2 induced Twist1 expression. Moreover, Sp1 transcription factor bound to the exon 1 CpG-rich region in Twist1 expression-positive cell lines, and Twist1 expression was diminished by mithramycin, which that interferes with Sp1 binding to CpG-rich regulatory sequences. Our studies suggested that the Twist1 transcription in GC cells might be regulated through potential cooperation of DNA methylation, histone modification in complex with Sp1 binding to CpG-rich regions within the exon 1 region. PMID:26695186

  14. Abnormal Methylation Status of the GNAS Exon 1A Region in Pseudohypohyperparathyroidism Combined With Turner Syndrome.

    PubMed

    Zhu, Jie; Wang, Dong; Ren, An; Xing, Yan; Zhang, Dongliang; Wei, Jun; Yu, Ning; Xing, Xuenong; Ye, Shandong

    2015-12-01

    Pseudohypohyperparathyroidism (PHHP) is a rare type of pseudohypoparathyroidism (PHP), which seems to have a normal skeletal response to parathyroid hormone but shows renal resistance. Almost all patients with PHHP have PHP Ib, a subtype of PHP that is usually caused by GNAS methylation defects, often in exon 1A. Some features of Albright hereditary osteodystrophy can occasionally be found in patients with PHHP, but these features are also common in Turner syndrome. The authors report on an extremely rare case of a patient with PHHP and Turner syndrome, a 47-year-old woman who sought medical attention for hypocalcemia and elevated parathyroid hormone. She had no family history of hypocalcemia and no STX16 gene deletions. She had a mosaic karyotype of 46, X, del(X)(p11.4)/45, XO. Pyrosequencing was performed to determine the GNAS exon 1A methylation. The degree of methylation found in exon 1A of the patient was lower than her unaffected relatives. PMID:26488942

  15. Promoting School Excellence through the Application of Effective Schools Research: Summary and Proceedings of a 1984 Regional Exchange Workshop (Nashville, Tennessee, April 15-16, 1984). Occasional Paper Series.

    ERIC Educational Resources Information Center

    Sattes, Beth D., Ed.

    A regional workshop was held in which educational researchers and practitioners shared their innovations, successes, concerns, and progress in using research and development to promote excellence in their state and local education agencies. This document reports its proceedings. "School Improvement: What the Research Says," by David P. Crandall,…

  16. Novel polymorphism in exon 1 of the melatonin receptor gene unassociated with reproductive characteristics of buffaloes in the Amazon Region.

    PubMed

    Barbosa, E M; Souza, B B; Guimarães, R C; Azevedo, J S N; Gonçalves, E C; Ribeiro, H F L; Rolim Filho, S T; Silva Filho, E

    2016-01-01

    The objective of this study was to sequence part of the exon 1 in the melatonin receptor 1A gene (MTRN1A) in buffaloes to detect a novel polymorphism with which to associate reproductive characteristics, such as age at first birth and the interval between births, in buffaloes from the northeastern region of the State of Pará (Brazil). Buffalo hair samples (77) were collected from the Terra Firme region of Pará. DNA was extracted and polymerase chain reactions (PCRs) were carried out with a primer that was designed using the GenBank accession No. AY524665 reference sequence. PCR products were purified and sequenced. After editing and analysis of the sequences, a mutation was observed at the 62nd position in exon 1 of MTRN1A (T↔C), which corresponded with a change in the 21st amino acid from leucine to proline. All possible genotypes were observed, with the most common being genotype CC (0.481). The allele frequencies were T = 0.377 and C = 0.623. Statistical analysis of FIS showed inbreeding within the sample group (FIS = 0.397) and deviations from the Hardy- Weinberg equilibrium were observed (P < 0.05). Associations between genotypes and reproductive characteristics were not significant (P > 0.05). Although the related SNP was not synonymous, there were no observable effects on the reproductive characteristics under investigation. As such, it would be ideal to detect other SNPs in exon 1 of the MTRN1A gene that can be associated with reproductive characteristics in Amazonian buffaloes. PMID:27421017

  17. Three indel variants in chicken LPIN1 exon 6/flanking region are associated with performance and carcass traits.

    PubMed

    Wang, R; Wang, T; Lu, W; Zhang, W; Chen, W; Kang, X; Huang, Y

    2015-01-01

    LPIN1 is a Mg(2+)-dependent phosphatidic acid phosphatase. Variation in chicken LPIN1 exon 6 and its flanking regions were identified and three indel variants in 6 breeds and their associations with performance traits were studied. Seven variants were detected from 6 breeds, which contained a synonymous tri-allelic variant (c.924A/T/C) and three indels. The exon 6 variants detected from chicken breeds were conserved among bird species. The indel variation frequency presented clear differences among breeds. Two coding indels (c.1014-1018del3 and c.1125-1138del12) were multiples of three nucleotides and maintained the open reading frames of LPIN1 proteins. However, they were predicted to result in the clear change of the RNA secondary structure of chicken LPIN1 exon 6 and LPIN1 protein conformation. The association analysis showed that c.871-15-22del6 variation had a significant effect on body weight at hatch (BW0) and 2 weeks (BW2); c. 1014-1018del3 variation had a significant effect on BW4, BW6, caecum length and gizzard weight (GW) traits; c.1125-1138del12 variation had a significant effect on BW12, shank length at 4 weeks (SL4), carcass weight, lactate dehydrogenase traits (LDH), glucose (GLU) and albumin (ALB) traits. The genotype combination for c.1014-1018del3 and c.1125-1138del12 also presented significant effects on SL4, SL8, GW, leg muscle weight, ALB, GLU and LDH. The study demonstrated that chicken LPIN1 has an important effect on body, carcass and organ weight, serum LDH, GLU and ALB level. PMID:26523976

  18. Three indel variants in chicken LPIN1 exon 6/flanking region are associated with performance and carcass traits.

    PubMed

    Wang, R; Wang, T; Lu, W; Zhang, W; Chen, W; Kang, X; Huang, Y

    2015-01-01

    LPIN1 is a Mg(2+)-dependent phosphatidic acid phosphatase. Variation in chicken LPIN1 exon 6 and its flanking regions were identified and three indel variants in 6 breeds and their associations with performance traits were studied. Seven variants were detected from 6 breeds, which contained a synonymous tri-allelic variant (c.924A/T/C) and three indels. The exon 6 variants detected from chicken breeds were conserved among bird species. The indel variation frequency presented clear differences among breeds. Two coding indels (c.1014-1018del3 and c.1125-1138del12) were multiples of three nucleotides and maintained the open reading frames of LPIN1 proteins. However, they were predicted to result in the clear change of the RNA secondary structure of chicken LPIN1 exon 6 and LPIN1 protein conformation. The association analysis showed that c.871-15-22del6 variation had a significant effect on body weight at hatch (BW0) and 2 weeks (BW2); c. 1014-1018del3 variation had a significant effect on BW4, BW6, caecum length and gizzard weight (GW) traits; c.1125-1138del12 variation had a significant effect on BW12, shank length at 4 weeks (SL4), carcass weight, lactate dehydrogenase traits (LDH), glucose (GLU) and albumin (ALB) traits. The genotype combination for c.1014-1018del3 and c.1125-1138del12 also presented significant effects on SL4, SL8, GW, leg muscle weight, ALB, GLU and LDH. The study demonstrated that chicken LPIN1 has an important effect on body, carcass and organ weight, serum LDH, GLU and ALB level.

  19. Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon.

    PubMed

    Lee, Cheng-Ming; Yen, Chia-Hung; Tzeng, Tsai-Yu; Huang, Yu-Zen; Chou, Kuan-Hsien; Chang, Tai-Jay; Arthur Chen, Yi-Ming

    2013-01-01

    Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer. PMID:23883094

  20. Origins and impacts of new mammalian exons

    PubMed Central

    Merkin, Jason; Chen, Ping; Alexis, Maria; Hautaniemi, Sampsa; Burge, Christopher B.

    2016-01-01

    Summary Mammalian genes are composed of exons, but the evolutionary origins and functions of new internal exons are poorly understood. Here, we analyzed patterns of exon gain using deep cDNA sequencing data from five mammals and one bird, identifying thousands of species- and lineage-specific exons. Most new exons derived from unique rather than repetitive intronic sequence. Unlike exons conserved across mammals, species-specific internal exons were mostly located in 5' untranslated regions and alternatively spliced. They were associated with upstream intronic deletions, increased nucleosome occupancy, and RNA polymerase II pausing. Genes containing new internal exons had increased gene expression, but only in tissues where the exon was included. Increased expression correlated with level of exon inclusion, promoter proximity, and signatures of cotranscriptional splicing. Together these findings suggest that splicing at the 5' ends of genes enhances expression and that changes in 5' end splicing alter gene expression between tissues and between species. PMID:25801031

  1. 46 CFR 193.15-16 - Lockout valves.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... EQUIPMENT Carbon Dioxide and Clean Agent Extinguishing Systems, Details § 193.15-16 Lockout valves. (a) A lockout valve must be provided on any carbon dioxide extinguishing system protecting a space over 6,000... complete isolation of the system from the protected space or spaces, making it impossible for...

  2. Alternative-Splicing in the Exon-10 Region of GABAA Receptor β2 Subunit Gene: Relationships between Novel Isoforms and Psychotic Disorders

    PubMed Central

    Zhao, Cunyou; Xu, Zhiwen; Wang, Feng; Chen, Jianhuan; Ng, Siu-Kin; Wong, Pak-Wing; Yu, Zhiliang; Pun, Frank W.; Ren, Lihuan; Lo, Wing-Sze; Tsang, Shui-Ying; Xue, Hong

    2009-01-01

    Background Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for β2-subunit of gamma-aminobutyric acid type A (GABAA) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing. Methods and Findings Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an “alternative splicing hotspot” that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, β2S1 and β2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased β2S1 expression and decreased β2S2 expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both β2S1 and β2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for β2S2 expression. Moreover, site-directed mutagenesis indicated that Thr365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. Conclusion This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to β2-subunit splicing diversity and the etiologies of SCZ and BPD. PMID:19763268

  3. Novel and recurrent BRCA2 mutations in Italian breast/ovarian cancer families widen the ovarian cancer cluster region boundaries to exons 13 and 14.

    PubMed

    Coppa, Anna; Buffone, Amelia; Capalbo, Carlo; Nicolussi, Arianna; D'Inzeo, Sonia; Belardinilli, Francesca; Colicchia, Valeria; Petroni, Marialaura; Granato, Teresa; Midulla, Cecilia; Zani, Massimo; Ferraro, Sergio; Screpanti, Isabella; Gulino, Alberto; Giannini, Giuseppe

    2014-12-01

    Hereditary breast and ovarian cancer are mainly linked to mutations in BRCA1 and BRCA2 genes which confer a similar cumulative risk of developing breast cancer. Importantly, while BRCA2 mutation carriers generally have a lower cumulative risk for ovarian cancer, mutations clustered in the central portion of BRCA2 are associated with a higher proportion of ovarian compared with breast cancer cases. The boundaries of this ovarian cancer cluster region (OCCR) have been tentatively defined within a 3.3 kb region of BRCA2 exon 11, and herein, we reassessed these boundaries using our series of Italian breast/ovarian cancer families. We used direct sequencing to investigate BRCA mutations in 367 breast/ovarian cancer families. We also studied the association between the location of the mutations and the ovarian cancer phenotype in our cohort of BRCA2-mutated families. We observed the novel c.7309_7309delA frameshift mutation and the c.7007G>A deleterious mutation in BRCA2 exons 14 and 13, respectively, in five independent Italian families characterized by a high proportion of ovarian cancer cases. Of note, a significantly higher proportion of ovarian versus breast cancer cases was associated not only with mutations in the previously defined OCCR (OR = 5.91; p = 0.004), but also with the exon 13-14 region (OR = 7.37; p = 0.001) in our BRCA2-mutated families. Our data provide initial evidence for a novel putative OCCR in BRCA2 exons 13-14.

  4. Red-shifting the optical response of firefly oxyluciferin with group 15/16 substitutions.

    PubMed

    Milne, Bruce F

    2014-12-01

    Time-dependent density functional theory has been used to investigate the effects of group 15/16 element substitution on the optical response of firefly oxyluciferin. A range of analogues containing symmetrical substitutions at the N and S atom positions of the naturally-occurring oxyluciferin have been found to have red-shifted electronic excitation energies with the heaviest derivative investigated (As/Se) displaying a shift of -0.69 eV. Fluorescence emission wavelengths for all P- and As-containing derivatives in DMSO are estimated to lie in the 710-930 nm region making them interesting for bio-imaging applications.

  5. Cleopatra Patera on Venus: Venera 15/16 evidence for a volcanic origin.

    USGS Publications Warehouse

    Schaber, G.G.; Kozak, R.C.; Masursky, H.

    1987-01-01

    The non-concentric nature, anomalous depth, and terraced morphology of the nested craters that compose Cleopatra Patera are more closely analogous to volcanic craters (calderas) than multi-ring impact structures. Associated deposits northeast and downslope of the Patera first recognized on Venera 15/16 radar images are interpreted as volcanic plains related to, and perhaps cogenetic with, Cleopatra. A volcanic origin not only is easily reconciled with the tectonic setting of the Patera, it is almost required by the correlation between the Patera and regional structural trend. -from Authors

  6. Genetic and physical mapping of 2q35 in the region of NRAMP and IL8R genes: Identification of a polymorphic repeat in exon 2 of NRAMP

    SciTech Connect

    White, J.K.; Shaw, M.A.; Barton, C.H.

    1994-11-15

    Recent interest has focused on the region of conserved synteny between mouse chromosome 1 and human 2q33-q37, particularly over the region encoding the murine macrophage resistance gene Ity/Lsh/Bcg (candidate Nramp) and members of the Il8r interleukin-8 (IL8) receptor gene cluster. In this paper, identification of a restriction fragment length polymorphism in the Il8RB gene in 35 pedigrees previously typed for markers in the 2q33-37 interval provided evidence (lod scores > 3) for linkage between Il8RB and the 2q34-135 markers FN1, TNP1, VIL1, and DES. Physical mapping, using yeast artificial chromosomes isolated with VIL1, confirmed that IL8RA, IL8RB and the IL8RB pseudogene map within the NRAMP-VIL1 interval, with the physical distance (155 kb) from 5{prime} LSH to 3{prime} VIL1 representing {approx}3-fold that observed in the mouse. Partial sequencing of NRAMP confirmed the presence of the N-terminal proline/serine-rich putative SH3 binding domain in exon 2 of the human gene. Further analysis of Brazilian leprosy and visceral leishmaniasis pedigrees identified a rare second allele varying in a 9-nucleotide repeat motif of the exon 2 sequence but segregating independently of the disease phenotype. 38 refs., 4 figs., 3 tabs.

  7. How are exons encoding transmembrane sequences distributed in the exon-intron structure of genes?

    PubMed

    Sawada, Ryusuke; Mitaku, Shigeki

    2011-01-01

    The exon-intron structure of eukaryotic genes raises a question about the distribution of transmembrane regions in membrane proteins. Were exons that encode transmembrane regions formed simply by inserting introns into preexisting genes or by some kind of exon shuffling? To answer this question, the exon-per-gene distribution was analyzed for all genes in 40 eukaryotic genomes with a particular focus on exons encoding transmembrane segments. In 21 higher multicellular eukaryotes, the percentage of multi-exon genes (those containing at least one intron) within all genes in a genome was high (>70%) and with a mean of 87%. When genes were grouped by the number of exons per gene in higher eukaryotes, good exponential distributions were obtained not only for all genes but also for the exons encoding transmembrane segments, leading to a constant ratio of membrane proteins independent of the exon-per-gene number. The positional distribution of transmembrane regions in single-pass membrane proteins showed that they are generally located in the amino or carboxyl terminal regions. This nonrandom distribution of transmembrane regions explains the constant ratio of membrane proteins to the exon-per-gene numbers because there are always two terminal (i.e., the amino and carboxyl) regions - independent of the length of sequences.

  8. 15 CFR 15.16 - Legal proceedings between private litigants: Expert or opinion testimony.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 1 2011-01-01 2011-01-01 false Legal proceedings between private litigants: Expert or opinion testimony. 15.16 Section 15.16 Commerce and Foreign Trade Office of the Secretary of Commerce LEGAL PROCEEDINGS Testimony by Employees and the Production of Documents in Legal Proceedings § 15.16 Legal proceedings...

  9. Cleopatra Patera on Venus - Venera 15/16 evidence for a volcanic origin

    NASA Astrophysics Data System (ADS)

    Schaber, G. G.; Kozak, R. C.; Masursky, H.

    1987-01-01

    The nonconcentric nature, anomalous depth, and terraced morphology of the nested craters that compose Cleopatra Patera are more closely analogous to volcanic craters (calderas) than multiring impact structures. Associated deposits northeast and downslope of the Patera first recognized on Venera 15/16 radar images are interpreted as volcanic plains related to, and perhaps cogenetic with, Cleopatra. The plains lavas probably originated as effusions from a radial rift zone or ring fissures. Rim deposits surrounding Cleopatra are asymmetric along the structural fabric of the region, also indicating fissure-type eruptions. Finally, a volcanic origin not only is easily reconciled with the tectonic setting of the Patera, it is almost required by the correlation between the Patera and regional structural trend.

  10. Results of reanalysis of the "Venera-9, 10, and 15, 16" bistatic data

    NASA Astrophysics Data System (ADS)

    Pavelyev, Alexander

    2012-07-01

    Insertion of the Venera-9,10 and -15,16 spacecraft into orbits around Venus affords the opportunity to make the bistatic and monostatic radar experiments. The main objective of these experiments is to study the topography and to measure the ground density of the planet. However, besides this objective, new data were obtained during the bistatic radar experiments on conditions of refraction of radio waves through the entire thickness of the atmosphere of Venus. The possibility of measuring refraction effects arose because the bistatic radar experiments were done immediately before the spacecraft passed into occultation by the planet. Therefore, the refraction effects were strongly evident. It should be noted also that the periodically repeated monostatic radar observations of Venus from Earth provided near the normal incidence direction give no information on refraction. The purpose of this contribution is to describe the method and the results of the experiments to measure the reflection properties of the surface and the refraction of radio waves in the atmosphere of Venus by means of bistatic radar. The author present measured values of the refractive angle and the coefficient of generalized spherical divergence of radio waves reflected from the surface of Venus. The measurements were made at wavelength 32 cm in the bistatic scheme using the Venera-9,10, and 15,16 spacecraft. Both the refraction angle and the reflection coefficient were determined in the experiment from the measured frequency difference between the direct and the reflected signals as a function of time, using the trajectory data on spacecraft positions. The reflection coefficient in the North Polar region varied considerably, which seems to correspond to significant variations in the conductivity of the soil. The results of the measurements agree with the theory of propagation of radio waves in the atmosphere of Venus. The work was partly supported by grant of Russian Fund of Basic Research No. 10

  11. Inhomogeneous DNA: Conducting exons and insulating introns

    NASA Astrophysics Data System (ADS)

    Krokhin, A. A.; Bagci, V. M. K.; Izrailev, F. M.; Usatenko, O. V.; Yampol'Skii, V. A.

    2009-08-01

    Parts of DNA sequences known as exons and introns play very different roles in coding and storage of genetic information. Here we show that their conducting properties are also very different. Taking into account long-range correlations among four basic nucleotides that form double-stranded DNA sequence, we calculate electron localization length for exon and intron regions. Analyzing different DNA molecules, we obtain that the exons have narrow bands of extended states, unlike the introns where all the states are well localized. The band of extended states is due to a specific form of the binary correlation function of the sequence of basic DNA nucleotides.

  12. Coincident indices of exons and introns.

    PubMed

    Xu, J; Chen, R; Ling, L; Shen, R; Sun, J

    1993-07-01

    In this paper, the coincident index, proposed by W. F. Friedman in cryptology, is made use of in DNA sequence analysis and exon prediction. The coincident index of exons exceeds that of introns by many times, and is mainly affected by window length, which is correlated negatively with the coincident index. An optimal exon prediction scheme was obtained by experimental analysis with an orthogonal table. Besides exons, many other special sites such as tandem repeats can be identified by using the coincident index approach. The application of this approach to the ARV-2 (AIDS associated retrovirus 2) genome found three new possible coding regions and some unusual base composition regions which are probably related to definite biological functions.

  13. Association between polymorphisms of exon 12 and exon 24 of JHDM2A gene and male infertility

    PubMed Central

    Hojati, Zohreh; Nouri Emamzadeh, Fatemeh; Dehghanian, Fariba

    2016-01-01

    Background: Some dynamic changes occurs during spermatogenesis such as histone removal and its replacement with transition nuclear protein and protamine. These proteins are required for packing and condensation of sperm chromatin. JHDM2A is a histone demethylase that directly binds to promoter regions of Tnp1 and Prm1 genes and controls their expression by removing H3K9 at their promoters. Objective: The association between polymorphisms of exon 12 and exon 24 in JHDM2A gene and male infertility were evaluated for the first time. Materials and Methods: In this experimental study, 400 infertile men (oligospermia and azoospermia) and normal healthy fathers were evaluated (n=200). Single Strand Conformation Polymorphism (SSCP-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were used for screening any polymorphisms that are exist in exon 12 and exon 24. Results: Exon 24 PCR products were analyzed by RFLP but no polymorphism was found in this exon at the restriction site of EcoRV enzyme. Our monitoring along the whole nucleotides of exon 12 and exon 24 were continued using SSCP method, but we found no change along these exons. Conclusion: Generally, this study evaluated the association between polymorphisms in exon 12 and exon 24 of JHDM2A gene and male infertility which suggests that polymorphisms of these exons may not be associated with the risk of male infertility. PMID:27525322

  14. Intronic motif pairs cooperate across exons to promote pre-mRNA splicing

    PubMed Central

    2010-01-01

    Background A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions. Results Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific. Conclusions The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition. PMID:20704715

  15. Dyslexia: Group Screening among 15-16-Year-Olds in Oslo, Norway

    ERIC Educational Resources Information Center

    Green, K.; Tonnessen, F. E.; Tambs, K.; Thoresen, M.; Bjertness, E.

    2009-01-01

    In 15-16 year olds from Oslo, Norway, we investigated the occurrence of self-reported dyslexia and reading/writing difficulties (RWD), and we measured dyslexic symptoms using the "Duvan" dyslexia screening test. The prevalence of self-reported dyslexia was 8.2%, while 10.4% reported severe or moderate RWD. The group of self-reported dyslexics…

  16. Papers from the Community College Governance Conference, February 15-16, 1974.

    ERIC Educational Resources Information Center

    Washington State Board for Community Coll. Education, Olympia.

    Papers presented at a conference on community college governance, held in Seattle, Washington on February 15-16, 1974, are provided. The papers are: "Governance for the Two-Year College" by Richard C. Richardson, Jr.: "The Faculty Stake in Governance" by Richard J. Frankie; "The Student Stake in Governance" by Alan R. Shark; "The Public Interest…

  17. 15 CFR 15.16 - Legal proceedings between private litigants: Expert or opinion testimony.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 15 Commerce and Foreign Trade 1 2013-01-01 2013-01-01 false Legal proceedings between private... Secretary of Commerce LEGAL PROCEEDINGS Testimony by Employees and the Production of Documents in Legal Proceedings § 15.16 Legal proceedings between private litigants: Expert or opinion testimony. In addition...

  18. 15 CFR 15.16 - Legal proceedings between private litigants: Expert or opinion testimony.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 15 Commerce and Foreign Trade 1 2012-01-01 2012-01-01 false Legal proceedings between private... Secretary of Commerce LEGAL PROCEEDINGS Testimony by Employees and the Production of Documents in Legal Proceedings § 15.16 Legal proceedings between private litigants: Expert or opinion testimony. In addition...

  19. 15 CFR 15.16 - Legal proceedings between private litigants: Expert or opinion testimony.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Legal proceedings between private... Secretary of Commerce LEGAL PROCEEDINGS Testimony by Employees and the Production of Documents in Legal Proceedings § 15.16 Legal proceedings between private litigants: Expert or opinion testimony. In addition...

  20. 15 CFR 15.16 - Legal proceedings between private litigants: Expert or opinion testimony.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 15 Commerce and Foreign Trade 1 2014-01-01 2014-01-01 false Legal proceedings between private... Secretary of Commerce LEGAL PROCEEDINGS Testimony by Employees and the Production of Documents in Legal Proceedings § 15.16 Legal proceedings between private litigants: Expert or opinion testimony. In addition...

  1. Promoter polymorphism T-786C, 894G→T at exon 7 of endothelial nitric oxide synthase gene are associated with risk of osteoporosis in Sichuan region male residents

    PubMed Central

    Gu, Zuchao; Zhang, Yu; Qiu, Guixing

    2015-01-01

    Objective: To investigate the association between genetic polymorphism of T-786C in promoter region, 894G→T at exon 7 of endothelial nitric oxide synthase (eNOS) gene and osteoporosis (OP) disease. Method: The genotypes of 350 patients with osteoporosis and 350 healthy controls were detected by polymerase chain reaction (PCR) and DNA sequencing. The allele ratios and genotype distributions in the patients and controls were assessed using the Pearson χ2-test. Odds ratios (OR) with two tailed P-values and 95% confidence intervals (CI) were calculated as a measure of the association of the eNOS genotypes with OP. Result: the C allele distribution frequency of T-786C eNOS gene in OP group (8.5%) was significantly higher than that in control group (3.9%), relative risk (OR) of OP associated with the CC genotype was 2.68 (95% CI, 0.92 to 1.37). The T allele frequency of 894G→T at exon 7 in eNOS gene in OP group (11.5%) was also significantly higher than that in control group (5.2%), OR of OP associated with the TT genotype was 2.60 (all P<0.05). Conclusion: The analysis results indicated that both T-786C in promoter region and 894G→T at exon 7 of eNOS gene might be genetic predisposal factors of OP, these polymorphisms may be independently or synergic with other loci to have an impact on the incidence of OP. PMID:26823879

  2. Cleopatra crater on Venus - Venera 15/16 data and impact/volcanic origin controversy

    NASA Astrophysics Data System (ADS)

    Basilevsky, A. T.; Ivanov, B. A.

    1990-02-01

    The morphology and morphometry of the 100-km diameter, 2.4-km deep Cleopatra crater on Venus are examined using Venera 15/16 images. The Cleopatra crater is compared to circular structures on Venus, Mercury, Mars, the earth and the moon. Consideration is given to the possible causes for the genesis of the Cleopatra crater. It is concluded that Cleopatra has a clear impact basin morphology with an anomalous crater depth.

  3. Ethnic group and alcohol consumption: the case of 15-16-year-olds in Leicestershire.

    PubMed

    Denscombe, M

    1995-03-01

    In recent years a number of research projects have reported on the level of alcohol use by young people. The research to date, however, has tended to overlook the ethnic origins of the young people as a factor associated with the level of alcohol use. Against this background, this paper reports findings on the use of alcohol by 15-16-year-olds in Leicestershire with specific reference to ethnic group. A survey of over 1000 young people revealed significant differences between ethnic groups in terms of their attitudes to alcohol consumption and the frequency of alcohol consumption. As expected, South Asians tended to hold less favourable attitudes to drinking alcohol than their White counterparts and reported a far lower frequency of alcohol consumption. The extent of the divergence between the two ethnic groups is detailed, revealing a stark contrast between the groups at age 15-16 years. Attitudes to alcohol consumption and reported frequency of alcohol consumption were also analysed in relation to the religion of the respondents. This analysis was undertaken to see if there were differences within the broader ethnic groups which would be disguised by aggregating the results under the headings of 'South Asian' and 'White'. Within the South Asian group, some differences in attitudes to the consumption of alcohol were found between Hindus, Sikhs and Muslims, with the Muslims exhibiting particular sensitivity to their religion's proscription of drinking alcohol. Despite this, the reported level of drinking by the Hindus and Sikhs was not much greater than that of the Muslims. The three groups tended to have a similar frequency of alcohol consumption which was markedly lower than that reported by the White 15-16-year-olds. The findings from the DART research have certain substantive and certain methodological implications in relation to epidemiological studies of alcohol consumption. Substantively, the findings reinforce the need to include ethnic group as a key

  4. Cryptic exon activation by disruption of exon splice enhancer: novel mechanism causing 3-methylcrotonyl-CoA carboxylase deficiency.

    PubMed

    Stucki, Martin; Suormala, Terttu; Fowler, Brian; Valle, David; Baumgartner, Matthias R

    2009-10-16

    3-Methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha (MCCA) and smaller beta (MCCB) subunits encoded by MCCA and MCCB, respectively. We report studies of the c.1054G-->A mutation in exon 11 of MCCB detected in the homozygous state in a patient with MCC deficiency. Sequence analysis of MCCB cDNA revealed two overlapping transcripts, one containing the normal 73 bp of exon 11 including the missense mutation c.1054G-->A (p.G352R), the other with exon 11 replaced by a 64-bp sequence from intron 10 (cryptic exon 10a) that maintains the reading frame and is flanked by acceptable splice consensus sites. In expression studies, we show that both transcripts lack detectable MCC activity. Western blot analysis showed slightly reduced levels of MCCB using the transcript containing the missense mutation, whereas no MCCB was detected with the transcript containing the cryptic exon 10a. Analysis of the region harboring the mutation revealed that the c.1054G-->A mutation is located in an exon splice enhancer sequence. Using MCCB minigene constructs to transfect MCCB-deficient fibroblasts, we demonstrate that the reduction in utilization of exon 11 associated with the c.1054G-->A mutation is due to alteration of this exon splice enhancer. Further, we show that optimization of the weak splice donor site of exon 11 corrects the splicing defect. To our knowledge, this is the first demonstration of a point mutation disrupting an exon splice enhancer that causes exon skipping along with utilization of a cryptic exon.

  5. Evolutionary history of exon shuffling.

    PubMed

    França, Gustavo S; Cancherini, Douglas V; de Souza, Sandro J

    2012-06-01

    Exon shuffling has been characterized as one of the major evolutionary forces shaping both the genome and the proteome of eukaryotes. This mechanism was particularly important in the creation of multidomain proteins during animal evolution, bringing a number of functional genetic novelties. Here, genome information from a variety of eukaryotic species was used to address several issues related to the evolutionary history of exon shuffling. By comparing all protein sequences within each species, we were able to characterize exon shuffling signatures throughout metazoans. Intron phase (the position of the intron regarding the codon) and exon symmetry (the pattern of flanking introns for a given exon or block of adjacent exons) were features used to evaluate exon shuffling. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the predominant kind in multicellular animals. Evidence is provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable presence of 1-1 shuffling units in Trichoplax adhaerens and a considerable prevalence of them in Nematostella vectensis. In contrast, Monosiga brevicollis, one of the closest relatives of metazoans, and Arabidopsis thaliana, showed no evidence of 1-1 exon or domain shuffling above what it would be expected by chance. Instead, exon shuffling events are less abundant and predominantly mediated by phase 0 introns (0-0 exon shuffling) in those non-metazoan species. Moreover, an intermediate pattern of 1-1 and 0-0 exon shuffling was observed for the placozoan T. adhaerens, a primitive animal. Finally, characterization of flanking intron phases around domain borders allowed us to identify a common set of symmetric 1-1 domains that have been shuffled throughout the metazoan lineage.

  6. Detection of 98. 5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene

    SciTech Connect

    Cuppens, H.; Marynen, P.; Cassiman, J.J. ); De Boeck, C. )

    1993-12-01

    The authors have previously shown that about 85% of the mutations in 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay. In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region and their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in the population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available. 24 refs., 1 fig., 2 tabs.

  7. A 2-nt RNA enhancer on exon 11 promotes exon 11 inclusion of the Ron proto-oncogene

    PubMed Central

    MOON, HEEGYUM; CHO, SUNGHEE; LOH, TIING JEN; ZHOU, JIANHUA; GHIGNA, CLAUDIA; BIAMONTI, GIUSEPPE; GREEN, MICHAEL R.; ZHENG, XUEXIU; SHEN, HAIHONG

    2014-01-01

    Ron is a human receptor for the macrophage-stimulating protein (MSP). Exon 11 skipping of Ron pre-mRNA produces the RonΔ165 protein that has a deletion of a 49 amino acid region in the β-chain extracellular domain. RonΔ165 is constitutively active even in the absence of its ligand. Through stepwise deletion analysis, we identified a 2-nt RNA enhancer, which is located 74 nt upstream from the 5′ splice site of exon 11, for exon 11 inclusion. Through double-base and single-base substitution analysis of the 2-nt RNA, we demonstrated that the GA, CC, UG and AC dinucleotides on exon 11, in addition to the wild-type AG sequence, function as enhancers for exon 11 inclusion of the Ron pre-mRNA. PMID:24189591

  8. Exon-intron organization and sequence comparison of human and murine T11 (CD2) genes

    SciTech Connect

    Diamond, D.J.; Clayton, L.K.; Sayre, P.H.; Reinherz, E.L.

    1988-03-01

    Genomic DNA clones containing the human and murine genes coding for the 50-kDa T11 (CD2) T-cell surface glycoprotein were characterized. The human T11 gene is approx. = 12 kilobases long and comprised of five exons. A leader exon (L) contains the 5'-untranslated region and most of the nucleotides defining the signal peptide (amino acids (aa) -24 to -5). Two exons encode the extracellular segment; exon Ex1 is 321 base pairs (bp) long and codes for four residues of the leader peptide and aa 1-103 of the mature protein, and exon Ex2 is 231 bp long and encodes aa 104-180. Exon TM is 123 bp long and codes for the single transmembrane region of the molecule (aa 181-221). Exon C is a large 765-bp exon encoding virtually the entire cytoplasmic domain (aa 222-327) and the 3'-untranslated region. The murine region T11 gene has a similar organization with exon-intron boundaries essentially identical to the human gene. Substantial conservation of nucleotide sequences between species in both 5'- and 3'-gene flanking regions equivalent to that among homologous exons suggests that murine and human genes may be regulated in a similar fashion. The probable relationship of the individual T11 exons to functional and structural protein domains is discussed.

  9. A genomic region encompassing a newly identified exon provides enhancing activity sufficient for normal myo7aa expression in zebrafish sensory hair cells.

    PubMed

    Ernest, Sylvain; Rosa, Frédéric M

    2015-09-01

    MYO7A is an unconventional myosin involved in the structural organization of hair bundles at the apex of sensory hair cells (SHCs) where it serves mechanotransduction in the process of hearing and balance. Mutations of MYO7A are responsible for abnormal shaping of hair bundles, resulting in human deafness and murine deafness/circling behavior. Myo7aa, expressed in SHCs of the inner ear and lateral line of zebrafish, causes circling behavior and abnormal hair cell function when deficient in mariner mutant. This work identifies a new hair cell-specific enhancer, highly conserved between species, located within Intron 2-3 of zebrafish myosin 7a (myo7aa) gene. This enhancer is contained within a 761-bp DNA fragment that encompasses a newly identified Exon of myo7aa and whose activity does not depend on orientation. Compensation of mariner mutation by expression of mCherry-Myo7aa fusion protein under the control of this 761-bp DNA fragment results in recovery of balance, normal hair bundle shape and restored hair cell function. Two smaller adjacent fragments (344-bp and 431-bp), extracted from the 761-bp fragment, both show hair cell-specific enhancing activity, with apparently reduced intensity and coverage. These data should help understand the role of Myo7aa in sensory hair cell differentiation and function. They provide tools to decipher how myo7aa gene is expressed and regulated in SHCs by allowing the identification of potential transcription factors involved in this process. The discovered enhancer could represent a new target for the identification of deafness-causing mutations affecting human MYO7A.

  10. A genomic region encompassing a newly identified exon provides enhancing activity sufficient for normal myo7aa expression in zebrafish sensory hair cells.

    PubMed

    Ernest, Sylvain; Rosa, Frédéric M

    2015-09-01

    MYO7A is an unconventional myosin involved in the structural organization of hair bundles at the apex of sensory hair cells (SHCs) where it serves mechanotransduction in the process of hearing and balance. Mutations of MYO7A are responsible for abnormal shaping of hair bundles, resulting in human deafness and murine deafness/circling behavior. Myo7aa, expressed in SHCs of the inner ear and lateral line of zebrafish, causes circling behavior and abnormal hair cell function when deficient in mariner mutant. This work identifies a new hair cell-specific enhancer, highly conserved between species, located within Intron 2-3 of zebrafish myosin 7a (myo7aa) gene. This enhancer is contained within a 761-bp DNA fragment that encompasses a newly identified Exon of myo7aa and whose activity does not depend on orientation. Compensation of mariner mutation by expression of mCherry-Myo7aa fusion protein under the control of this 761-bp DNA fragment results in recovery of balance, normal hair bundle shape and restored hair cell function. Two smaller adjacent fragments (344-bp and 431-bp), extracted from the 761-bp fragment, both show hair cell-specific enhancing activity, with apparently reduced intensity and coverage. These data should help understand the role of Myo7aa in sensory hair cell differentiation and function. They provide tools to decipher how myo7aa gene is expressed and regulated in SHCs by allowing the identification of potential transcription factors involved in this process. The discovered enhancer could represent a new target for the identification of deafness-causing mutations affecting human MYO7A. PMID:25556989

  11. Widespread Shortening of 3’ Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection

    PubMed Central

    Pagé Sabourin, Ariane; Nédélec, Yohann; Dumaine, Anne; Yotova, Vania; Johnson, Zachary P.; Lanford, Robert E.; Burge, Christopher B.

    2016-01-01

    The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3’ UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses. PMID:27690314

  12. Pervasive Layering in the Lunar Highland Crust: Evidence from Apollos 15, 16,and 17

    NASA Technical Reports Server (NTRS)

    Lowman, Paul D., Jr.; Yang, Tiffany

    2005-01-01

    This paper presents results of a photogeologic reconnaissance of 70 mm photographs taken on the lunar surface during the Apollo 15, 16, and 17 missions, whose primary objective was to investigate the lunar highland crust. Photographs at all three sites, notably the Apennine Front, show pervasive layered structure. These layers are easily distinguished from lighting artifacts, and are considered genuine crustal structures. Their number, thickness, and extent implies that they are lava flows, not ejecta blankets or intrusive features. They appear to be the upper part of the earliest lunar crust, possibly forming a layer tens of kilometers thick. Remote sensing studies (X-ray fluorescence and reflectance spectroscopy), indicate that the highland crust is dominantly a feldspathic basalt. It is concluded that the highland layers represent a global crust formed by eruptions of high-alumina basalt in the first few hundred million years of the Moon's history.

  13. Intravenous kinetics and metabolism of [15,16-3H]naltrexonium methiodide in the rat.

    PubMed

    Misra, A L; Pontani, R B; Vadlamani, N L

    1987-03-01

    After a 4 mg kg-1 bolus intravenous dose of [15,16-3H]naltrexonium methiodide to the rat, brain to plasma concentration ratios of the compound were 0.031 to 0.228 between 0.25 to 6 h after injection and the t 1/2 beta in plasma and brain were 2.92 and 7.61 h, respectively. Ethyl acetate-extracted radioactivity due to metabolites in plasma decayed with t 1/2 beta 1.83 h and the ratios of plasma concentration of metabolites to quaternary compound between 0.25 and 6 h were 0.014-0.026. Only unconjugated 7,8-dihydro-14-hydroxynormorphine, naltrexone and traces of 7,8-dihydro-14-hydroxynormorphinone were the metabolites in plasma. Naltrexone (but not normetabolites) was present only in traces in brain up to 0.5 h after injection and not at later times. PMID:2883290

  14. Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

    PubMed

    Quinones, S; Bernal, D; García-Sogo, M; Elena, S F; Saus, J

    1992-10-01

    The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.

  15. Exonal deletion of SLC24A4 causes hypomaturation amelogenesis imperfecta.

    PubMed

    Seymen, F; Lee, K-E; Tran Le, C G; Yildirim, M; Gencay, K; Lee, Z H; Kim, J-W

    2014-04-01

    Amelogenesis imperfecta is a heterogeneous group of genetic conditions affecting enamel formation. Recently, mutations in solute carrier family 24 member 4 (SLC24A4) have been identified to cause autosomal recessive hypomaturation amelogenesis imperfecta. We recruited a consanguineous family with hypomaturation amelogenesis imperfecta with generalized brown discoloration. Sequencing of the candidate genes identified a 10-kb deletion, including exons 15, 16, and most of the last exon of the SLC24A4 gene. Interestingly, this deletion was caused by homologous recombination between two 354-bp-long homologous sequences located in intron 14 and the 3' UTR. This is the first report of exonal deletion in SLC24A4 providing confirmatory evidence that the function of SLC24A4 in calcium transport has a crucial role in the maturation stage of amelogenesis.

  16. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies.

    PubMed

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M; Krishnaswarmy, Sudarsan; Wong, Brenda L; Fletcher, Sue; Wilton, Steve D

    2016-01-01

    Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes. PMID:26745801

  17. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

    PubMed Central

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M.; Krishnaswarmy, Sudarsan; Wong, Brenda L.; Fletcher, Sue; Wilton, Steve D.

    2016-01-01

    Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes. PMID:26745801

  18. Exonization of the LTR transposable elements in human genome

    PubMed Central

    Piriyapongsa, Jittima; Polavarapu, Nalini; Borodovsky, Mark; McDonald, John

    2007-01-01

    Background Retrotransposons have been shown to contribute to evolution of both structure and regulation of protein coding genes. It has been postulated that the primary mechanism by which retrotransposons contribute to structural gene evolution is through insertion into an intron or a gene flanking region, and subsequent incorporation into an exon. Results We found that Long Terminal Repeat (LTR) retrotransposons are associated with 1,057 human genes (5.8%). In 256 cases LTR retrotransposons were observed in protein-coding regions, while 50 distinct protein coding exons in 45 genes were comprised exclusively of LTR RetroTransposon Sequence (LRTS). We go on to reconstruct the evolutionary history of an alternatively spliced exon of the Interleukin 22 receptor, alpha 2 gene (IL22RA2) derived from a sequence of retrotransposon of the Mammalian apparent LTR retrotransposons (MaLR) family. Sequencing and analysis of the homologous regions of genomes of several primates indicate that the LTR retrotransposon was inserted into the IL22RA2 gene at least prior to the divergence of Apes and Old World monkeys from a common ancestor (~25 MYA). We hypothesize that the recruitment of the part of LTR as a novel exon in great ape species occurred prior to the divergence of orangutans and humans from a common ancestor (~14 MYA) as a result of a single mutation in the proto-splice site. Conclusion Our analysis of LRTS exonization events has shown that the patterns of LRTS distribution in human exons support the hypothesis that LRTS played a significant role in human gene evolution by providing cis-regulatory sequences; direct incorporation of LTR sequences into protein coding regions was observed less frequently. Combination of computational and experimental approaches used for tracing the history of the LTR exonization process of IL22RA2 gene presents a promising strategy that could facilitate further studies of transposon initiated gene evolution. PMID:17725822

  19. Quantitative analysis of terrain units mapped in the northern quarter of Venus from Venera 15/16 data

    NASA Technical Reports Server (NTRS)

    Schaber, G. G.

    1991-01-01

    The contacts between 34 geological/geomorphic terrain units in the northern quarter of Venus mapped from Venera 15/16 data were digitized and converted to a Sinusoidal Equal-Area projection. The result was then registered with a merged Pioneer Venus/Venera 15/16 altimetric database, root mean square (rms) slope values, and radar reflectivity values derived from Pioneer Venus. The resulting information includes comparisons among individual terrain units and terrain groups to which they are assigned in regard to percentage of map area covered, elevation, rms slopes, distribution of suspected craters greater than 10 km in diameter.

  20. Exon trapping of internal and 3{prime}-terminal exons from a YAC containing the AML1 gene

    SciTech Connect

    Ally, A.; Nisson, P.E.

    1994-09-01

    The t(8;21) translocation is associated with a high percentage of acute myelogenous leukemia (AML) cases of type 2 FAB. This cytogenetic landmark has been instrumental in the positional cloning of the AML1 gene which encodes a transcription factor and spans the translocation region. Using 3{prime} RACE and exon trapping, multiple AML1 transcripts have been observed which are generated by alternative splicing 3{prime} to exon 5. Although several transcripts from the AML1 gene have been cloned, these account for only a fraction of those predicted by Northern blotting. We therefore have subjected a 240 kb YAC (C4C10) that contains the entire AML1 gene to internal and 3{prime}-terminal exon trapping in an attempt to fully characterize the transcript repetoire from AML1. Exon trapping has been shown previously to capture exonic sequence by selecting splicing signals and has been applied primarily on cosmids. We report here the development of protocols for the efficient capture of internal and 3{prime}-terminal exons from the AML1 gene directly from YAC DNA.

  1. The New Outlook for Science. Science and Belief: from Copernicus to Darwin, Block VI, Units 15-16.

    ERIC Educational Resources Information Center

    Open Univ., Walton, Bletchley, Bucks (England).

    This text contains units 15-16 in the Open University course, Science and Belief: from Copernicus to Darwin. It is an inter-faculty second level course in the history of science. Unit 15 is concerned with Nature and History and includes uniformitarianism, human history, evolutionism, and Darwinism. Unit objectives, readings, and questions with the…

  2. A bicyclic diterpenoid with a new 15,16-dinorlabdane carbon skeleton from Leonurus japonicus and its coagulant bioactivity.

    PubMed

    Peng, Fu; Xiong, Liang; Zhao, Xiao-Mei

    2013-01-01

    A new 15,16-dinorlabdane diterpenoid 1 and a known labdane diterpenoid 2, together with three known ergosterols 3-5, were isolated from the EtOAc-soluble portion of the EtOH extract of Leonurus japonicus. Their structures were elucidated by physical and spectroscopic analysis. Compound 1 showed in vitro coagulant activity in the APTT, PT, TT, and FIB assays.

  3. Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro.

    PubMed Central

    Chan, R C; Black, D L

    1995-01-01

    The neuron-specific N1 exon of the mouse c-src transcript is normally skipped in nonneuronal cells. In this study, we examined the sequence requirements for the exclusion of this exon in nonneuronal HeLa cell nuclear extracts. We found that the repression of the N1 exon is mediated by specific intron sequences that flank the N1 exon. Mutagenesis experiments identified conserved CUCUCU elements within these intron regions that are required for the repression of N1 splicing. The addition of an RNA competitor containing the upstream regulatory sequence to the HeLa extract induced splicing of the intron downstream of N1, indicating that the competitor sequence binds to splicing repressor proteins. The similarities between this mechanism for src splicing repression and the repression of other regulated exons point to a common role of exon-spanning interactions in splicing repression. PMID:7565790

  4. Exon structure of the human dystrophin gene

    SciTech Connect

    Roberts, R.G.; Coffey, A.J.; Bobrow, M.; Bentley, D.R.

    1993-05-01

    Application of a novel vectorette PCR approach to defining intron-exon boundaries has permitted completion of analysis of the exon structure of the largest and most complex known human gene. The authors present here a summary of the exon structure of the entire human dystrophin gene, together with the sizes of genomic HindIII fragments recognized by each exon, and (where available) GenBank accession numbers for adjacent intron sequences. 20 refs., 1 tab.

  5. Recombinant Exon-Encoded Resilins for Elastomeric Biomaterials

    PubMed Central

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Arinus, Shira B.; Dgany, Or; Shoseyov, Oded; Kaplan, David L.

    2011-01-01

    Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediateed dityrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative dityrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in-vivo applications of resilin biomaterials. PMID:21963157

  6. SOX11 MODULATES BRAIN-DERIVED NEUROTROPHIC FACTOR EXPRESSION IN AN EXON PROMOTER-SPECIFIC MANNER

    PubMed Central

    Salerno, Kathleen M.; Jing, Xiaotang; Diges, Charlotte M.; Cornuet, Pamela K.; Glorioso, Joseph C.; Albers, Kathryn M.

    2011-01-01

    Sox11 is a high mobility group (HMG) containing transcription factor that is significantly elevated in peripheral neurons in response to nerve injury. In vitro and in vivo studies support a central role for Sox11 in adult neuron growth and survival following injury. Brain-derived neurotrophic factor (BDNF) is a pleiotropic growth factor that has effects on neuronal survival, differentiation, synaptic plasticity and regeneration. BDNF transcription is elevated in the DRG following nerve injury in parallel with Sox11 allowing for the possible regulation by Sox11. To begin to assess the possible influence of Sox11 we used reverse transcriptase PCR assays to determine the relative expression of the nine (I-IXa) noncoding exons and one coding exon (exon IX) of the BDNF gene after sciatic nerve axotomy in the mouse. Exons with upstream promoter regions containing the Sox binding motif 5′-AACAAAG-3′ (I, IV, VII and VIII) were increased at 1d or 3d following axotomy. Exons 1 and IV showed the greatest increase and only exon 1 remained elevated at 3d. Luciferase assays showed that Sox11 could activate the most highly regulated exons, I and IV, and that this activation was reduced by mutation of putative Sox binding sites. Exon expression in injured DRG neurons had some overlap with Neuro2a cells that overexpress Sox11, showing elevation in exon IV and VII transcripts. These findings indicate cell type and contextual specificity of Sox11 in modulation of BDNF transcription. PMID:22331573

  7. Conserved sequence elements associated with exon skipping

    PubMed Central

    Miriami, Elana; Margalit, Hanah; Sperling, Ruth

    2003-01-01

    One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5′ and 3′ splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping. PMID:12655015

  8. Applications of artificial intelligence IV; Proceedings of the Meeting, Innsbruck, Austria, Apr. 15, 16, 1986

    SciTech Connect

    Gilmore, J.F.

    1986-01-01

    An expert system for roving robotics; hybrid expert systems in image analysis; the use of PROLOG in automatic speech recognition; a knowledge-based geological prospecting system; the use of AI technology in failsafe real-time systems; computer vision for artificially intelligent robotic systems; and a knowledge representation system for searching trajectories in robotics are examined. Attention is given to using contextual data in classification algorithms; real-time intelligent hardware-based image processing; a parallel intelligent system; a LISP machine; a graphical display intelligence software based on a raster scan; and the extraction of uniform regions with minimization of noise points effects. Topics discussed include an active coordinate imaging system for robot vision; the design of a smart sensor system for real-time remote sensing image processing on-board a satellite; texture defects and visual inspection; the three-dimensional modeling of industrial parts for image analysis; and a new type of cellular turing acceptor.

  9. Exon circularization in mammalian nuclear extracts.

    PubMed

    Pasman, Z; Been, M D; Garcia-Blanco, M A

    1996-06-01

    Correct ligation of exons in pre-mRNA splicing requires splice site juxtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice site, however, is predicted to result in a circular RNA. This mode of splice site pairing across the axon has been hypothesized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site pairing has been postulated to explain the formation of SRY circular transcripts in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that splice site pairing across the exon can result in exon circularization in vitro. These results indicate that spliceosome-mediated axon circularization indeed can account for the formation of scrambled exons and circular RNAs. Exon circularization efficiency decreased dramatically as the length of the exon was increased from 95 nt to 274 nt. Circularization of this longer exon was restored, however, when intronic complementary sequences were included in the RNA substrate. These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing. Therefore, the splicing of this structured RNA recapitulated SRY-like exon circularization in vitro.

  10. CoNVaDING: Single Exon Variation Detection in Targeted NGS Data.

    PubMed

    Johansson, Lennart F; van Dijk, Freerk; de Boer, Eddy N; van Dijk-Bos, Krista K; Jongbloed, Jan D H; van der Hout, Annemieke H; Westers, Helga; Sinke, Richard J; Swertz, Morris A; Sijmons, Rolf H; Sikkema-Raddatz, Birgit

    2016-05-01

    We have developed a tool for detecting single exon copy-number variations (CNVs) in targeted next-generation sequencing data: CoNVaDING (Copy Number Variation Detection In Next-generation sequencing Gene panels). CoNVaDING includes a stringent quality control (QC) metric, that excludes or flags low-quality exons. Since this QC shows exactly which exons can be reliably analyzed and which exons are in need of an alternative analysis method, CoNVaDING is not only useful for CNV detection in a research setting, but also in clinical diagnostics. During the validation phase, CoNVaDING detected all known CNVs in high-quality targets in 320 samples analyzed, giving 100% sensitivity and 99.998% specificity for 308,574 exons. CoNVaDING outperforms existing tools by exhibiting a higher sensitivity and specificity and by precisely identifying low-quality samples and regions.

  11. Contrasting chromatin organization of CpG islands and exons in the human genome

    PubMed Central

    2010-01-01

    Background CpG islands and nucleosome-free regions are both found in promoters. However, their association has never been studied. On the other hand, DNA methylation is absent in promoters but is enriched in gene bodies. Intragenic nucleosomes and their modifications have been recently associated with RNA splicing. Because the function of intragenic DNA methylation remains unclear, I explored the possibility of its involvement in splicing regulation. Results Here I show that CpG islands were associated not only with methylation-free promoters but also with nucleosome-free promoters. Nucleosome-free regions were observed only in promoters containing a CpG island. However, the DNA sequences of CpG islands predicted the opposite pattern, implying a limitation of sequence programs for the determination of nucleosome occupancy. In contrast to the methylation-and nucleosome-free states of CpG-island promoters, exons were densely methylated at CpGs and packaged into nucleosomes. Exon-enrichment of DNA methylation was specifically found in spliced exons and in exons with weak splice sites. The enrichment patterns were less pronounced in initial exons and in non-coding exons, potentially reflecting a lower need for their splicing. I also found that nucleosomes, DNA methylation, and H3K36me3 marked the exons of transcripts with low, medium, and high gene expression levels, respectively. Conclusions Human promoters containing a CpG island tend to remain nucleosome-free as well as methylation-free. In contrast, exons demonstrate a high degree of methylation and nucleosome occupancy. Exonic DNA methylation seems to function together with exonic nucleosomes and H3K36me3 for the proper splicing of transcripts with different expression levels. PMID:20602769

  12. ExonMiner: Web service for analysis of GeneChip Exon array data

    PubMed Central

    Numata, Kazuyuki; Yoshida, Ryo; Nagasaki, Masao; Saito, Ayumu; Imoto, Seiya; Miyano, Satoru

    2008-01-01

    Background Some splicing isoform-specific transcriptional regulations are related to disease. Therefore, detection of disease specific splice variations is the first step for finding disease specific transcriptional regulations. Affymetrix Human Exon 1.0 ST Array can measure exon-level expression profiles that are suitable to find differentially expressed exons in genome-wide scale. However, exon array produces massive datasets that are more than we can handle and analyze on personal computer. Results We have developed ExonMiner that is the first all-in-one web service for analysis of exon array data to detect transcripts that have significantly different splicing patterns in two cells, e.g. normal and cancer cells. ExonMiner can perform the following analyses: (1) data normalization, (2) statistical analysis based on two-way ANOVA, (3) finding transcripts with significantly different splice patterns, (4) efficient visualization based on heatmaps and barplots, and (5) meta-analysis to detect exon level biomarkers. We implemented ExonMiner on a supercomputer system in order to perform genome-wide analysis for more than 300,000 transcripts in exon array data, which has the potential to reveal the aberrant splice variations in cancer cells as exon level biomarkers. Conclusion ExonMiner is well suited for analysis of exon array data and does not require any installation of software except for internet browsers. What all users need to do is to access the ExonMiner URL . Users can analyze full dataset of exon array data within hours by high-level statistical analysis with sound theoretical basis that finds aberrant splice variants as biomarkers. PMID:19036125

  13. Human decorin gene: Intron-exon junctions and chromosomal localization

    SciTech Connect

    Vetter, U.; Young, M.F.; Fisher, L.W. ); Vogel, W.; Just, W. )

    1993-01-01

    All of the protein-encoding exons and the 3[prime]flanking region of the human decorin gene have been cloned an partially sequenced. The locations of the intron-exon junctions within the coding portion of the gene were identical to those found for the homologous human gene, biglycan. The sizes of the introns in the decorin gene, however, were substantially larger than those of the same introns of the biglycan gene. Portions of introns 1, 2, and 3 as well as exon 1 were not found during our extensive screening process. The 5[prime] end of intron 2 was found to have an AG-rich region followed immediately by a CT-rich region. Furthermore, the 5[prime] end of intron 3 was very rich in thymidine, whereas the 3[prime] end of intron 7 was rich in adenosine. Several cDNA clones constructed from cultured human bone cell mRNA were found to contain a different sequence at the 5[prime] end compared to that previously published for mRNA from a human embryonic fibroblast cell line. We were also unable to find the alternate 3[prime] flanking region of the previously published cDNA sequence. We have mapped the human decorin gene by in situ methods to chromosome 12q2l.3. 30 refs., 3 figs., 1 tab.

  14. 11p15-subband specific search for transcribed sequences using exon trapping

    SciTech Connect

    Loebbert, R.; Prawitt, D.; Monroe, D.

    1994-09-01

    Evidence from cytogenetic and molecular data suggest that the region 11p15 contains genes involved in different disorders, like Beckwith-Wiedemann syndrome (BWS), long QT syndrome (LQT), Usher syndrome type I and tumor development. Focusing on the subregion 11p15.1, we are isolating and characterizing new transcribed sequences. The applied strategy includes exon amplification and subsequent PCR screening of cDNA libraries. So far 100 YACs and 38 cosmid clones from 11p15.1-15.3 have been collected and are currently arrayed. 16 cosmids have been analyzed for transcribed sequences using the exon amplification scheme developed by Buckler et al. (1991). We were able to identify 18 exons that contain correct open reading frames and map back to the cosmid clones. A data base search revealed that two exons represent parts of known genes from this region (ST5 and AMPD3). Moreover, we identified one exon that represents an EGF-like repeat with homologies to various proteins. Using PCR and primers from the exon sequences, a fetal brain library, which has been arranged in the form of hierarchic arrayed phage pools, was screened. Up to now, two cDNA clones corresponding to different exons were isolated and are currently sequenced.

  15. The Contribution of Exon-Skipping Events on Chromosome 22 to Protein Coding Diversity

    PubMed Central

    Hide, Winston A.; Babenko, Vladimir N.; van Heusden, Peter A.; Seoighe, Cathal; Kelso, Janet F.

    2001-01-01

    Completion of the human genome sequence provides evidence for a gene count with lower bound 30,000–40,000. Significant protein complexity may derive in part from multiple transcript isoforms. Recent EST based studies have revealed that alternate transcription, including alternative splicing, polyadenylation and transcription start sites, occurs within at least 30–40% of human genes. Transcript form surveys have yet to integrate the genomic context, expression, frequency, and contribution to protein diversity of isoform variation. We determine here the degree to which protein coding diversity may be influenced by alternate expression of transcripts by exhaustive manual confirmation of genome sequence annotation, and comparison to available transcript data to accurately associate skipped exon isoforms with genomic sequence. Relative expression levels of transcripts are estimated from EST database representation. The rigorous in silico method accurately identifies exon skipping using verified genome sequence. 545 genes have been studied in this first hand-curated assessment of exon skipping on chromosome 22. Combining manual assessment with software screening of exon boundaries provides a highly accurate and internally consistent indication of skipping frequency. 57 of 62 exon skipping events occur in the protein coding regions of 52 genes. A single gene, (FBXO7) expresses an exon repetition. 59% of highly represented multi-exon genes are likely to express exon-skipped isoforms in ratios that vary from 1:1 to 1:>100. The proportion of all transcripts corresponding to multi-exon genes that exhibit an exon skip is estimated to be 5%. PMID:11691849

  16. Haplotype diversity in the human red and green opsin genes: evidence for frequent sequence exchange in exon 3.

    PubMed

    Winderickx, J; Battisti, L; Hibiya, Y; Motulsky, A G; Deeb, S S

    1993-09-01

    We studied polymorphisms in the coding sequences of the human red and green opsin genes of 133 Caucasian males. Eleven polymorphic sites were discovered in the red opsin gene, seven of which were in exon 3, three in exon 4 and one in exon 5. Polymorphisms at 8 of these sites resulted in amino acid substitutions which generated a total of 18 unique red opsins in the population. The substitutions at three (S180A, I230T, and A233S) of the 8 sites involve hydroxyl-bearing to non-polar amino acid residues, and are therefore likely to alter spectral characteristics of the red pigment. Eight polymorphic sites were observed in the green opsin coding sequences, six of which were in exon 3, one in exon 2 and one in exon 5. Five of the eight involved amino acid substitutions which generated 15 unique green opsins in the population. Substitutions at two of these sites involve hydroxyl-bearing vs. non-polar residues. Six polymorphisms, all of which are located in exon 3, are shared between the red and green opsin genes, essentially making it difficult to assign this exon to either of these genes. Markers in exon 3 are in partial linkage disequilibrium with those in exons 4 and 5, whereas the latter two are in strong linkage disequilibrium with each other. Furthermore, markers in the 5' region of exon 3 are also in only partial (54%) disequilibrium with those in the 3' region. The above results strongly suggest a history of frequent gene conversion, mainly localized to exon 3, in the lineages leading to the human red and green opsin genes.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat

    PubMed Central

    Khichar, Jai Prakash; Gahlot, Gyan Chand; Agrawal, Vijay Kumar; Kiran; Dewna, Ajay Singh; Prakash; Ashraf, Mohammad

    2016-01-01

    Aim: To estimate genetic variability in exon 3 of caprine myostatin gene in Marwari goats. Materials and Methods: A total of 120 blood samples from unrelated Marwari goats were randomly collected from different villages of Bikaner (Rajasthan), India. Genomic DNA was extracted from whole blood using blood DNA isolation kit (Himedia Ltd.) as per manufacturer’s protocol. The quality of extracted genomic DNA was checked on 0.8% agarose gel. Specifically designed a primer set for caprine myostatin (MSTN) gene (Genebank accession no. DQ167575) was used to amplify the exon 3 region of MSTN gene in Marwari goat. The genetic variability in exon 3 of MSTN gene in Marwari goat was assessed on 8% polyacrylamide gel electrophoresis to detect single strand conformation polymorphism (SSCP) pattern. Results: The exon 3 of MSTN gene in Marwari goat showed two types of conformation patterns on 8% polyacrylamide gel. One of the patterns showed only two bands and was considered as genotype AA, whereas another pattern having an extra band was designated as genotype AB. The frequencies of AA and AB genotype for exon 3 region of MSTN gene were calculated as 0.90 and 0.10, respectively. Conclusion: Low level of polymorphism was observed at exon 3 region of MSTN gene in Marwari goat through SSCP analysis. This information could be utilized in future breeding plan to exploit the unique characteristics of Marwari goat of Rajasthan. PMID:27397994

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

    SciTech Connect

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

    1994-09-01

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

  19. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

  20. Low power consumption high speed CMOS dual-modulus 15/16 prescaler for optical and wireless communications

    NASA Astrophysics Data System (ADS)

    Liu, Hui-Min; Zhang, Xiao-Xing; Dai, Yu-Jie; Lv, Ying-Jie

    2011-09-01

    Frequency synthesizer is an important part of optical and wireless communication system. Low power comsumption prescaler is one of the most critical unit of frequency synthesizer. For the frequency divider, it must be programmable for channel selection in multi-channel communication systems. A dual-modulus prescaler (DMP) is needed to provide variable division ratios. DMP is considered as a critical power dissipative block since it always operates at full speed. This paper introduces a high speed and low power complementary metal oxide semiconductor (CMOS) 15/16 DMP based on true single-phase-clock (TSPC) and transmission gates (TGs) cell. A conventional TSPC is optimized in terms of devices size, and it is resimulated. The TSPC is used in the synchronous and asynchronous counter. TGs are used in the control logic. The DMP circuit is implemented in 0.18 μm CMOS process. The simulation results are provided. The results show wide operating frequency range from 7.143 MHz to 4.76 GHz and it comsumes 3.625 mW under 1.8 V power supply voltage at 4.76 GHz.

  1. High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

    PubMed Central

    Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

    2009-01-01

    JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

  2. Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta.

    PubMed

    Wilkie, Hazel; Xu, Siyang; Gossner, Anton; Hopkins, John

    2015-09-15

    The resistance and susceptibility of sheep to the common abomasal nematode parasite, Teladorsagia circumcincta is strongly associated with the differential polarization of the immune response. Resistant animals control larval colonization by the production of a protective antibody response regulated by Th2 T cells. Susceptible sheep respond to infection by developing an inflammatory Th1/Th17 response that fails to control infection. Previous microarray analysis identified genes associated with T cell polarization that were differentially expressed between the resistant and susceptible sheep. RT-qPCR confirmed the microarray data for ALOX15 and IL13. Both ALOX15 exon 9 and IL13 exon 4 were significantly increased in resistant animals and copy number RT-qPCR showed that expression levels of these exons were significantly negatively correlated with quantitative phenotypic traits, including abomasal worm counts and faecal egg counts. Sequencing of the intronic regions 5' to these genes failed to identify any potential genetic links to differential exon usage.

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

    SciTech Connect

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

    1989-02-01

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

  4. The complete sequence of the human CD79b (Ig{beta}/B29) gene: Identification of a conserved exon/intron organization, immunoglobulin-like regulatory regions, and allelic polymorphism

    SciTech Connect

    Hashimoto, S.; Chiorazzi, N.; Gregersen, P.K. |

    1994-12-31

    We determined the complete genomic sequence of the human CD79b (Ig{beta}/B29) gene. The CD79b gene product is associated with the membrane immunoglobulin signaling complex which is composed of immunoglobulin (Ig) itself, associated in a noncovalent fashion with CD79b and a second polypeptide chain, CD79a (Ig{alpha}/mb1). The sequence and exon/intron organization of the human and mouse CD79b genes are highly similar. The gene organization suggests that some variant forms of CD79b may arise by virtue of alternative splicing of mRNA. In addition, a number of conserved regulatory sequences commonly found in Ig genes are present in sequences which flank the human CD79b gene. Some of these sequences are distinct from those found in the CD79a promoter. These differences may explain why transcription of CD79b, but not CD79a, is observed in plasma cells. A new Taq 1 restriction fragment length polymorphism is described that is not associated with any structural polymorphisms of the expressed CD79b polypeptide. 13 refs., 3 figs., 1 tab.

  5. Parachuting injuries during Operation Royal Dragon, Big Drop III, Fort Bragg, North Carolina, May 15/16, 1996.

    PubMed

    Craig, S C; Zugner, D; Knapik, J J; Bricknell, M C

    1999-01-01

    On the night of May 15/16, 1996, the largest parachute assault of United States (US) and United Kingdom (UK) airborne forces in 52 years occurred at Fort Bragg, North Carolina. This paper describes the injuries sustained in that operation. A total of 4,754 (US, N = 3,066; UK, N = 1,688) aircraft exits were made, causing a total of 137 (US, N = 73; UK, N = 64) injuries in 117 personnel (US = 68; UK = 49). There were 15 hospital admissions (US = 8; UK = 7; p = 0.37) and no fatalities. The combined exit injury incidence was 24.6 injured soldiers per 1,000 exits. The US exit injury rate was 22 injured per 1,000 aircraft exits and the UK rate was 29 injured soldiers per 1,000 aircraft exits. This difference was not statistically significant (p = 0.25). Lower extremity sprains, strains, and fractures accounted for the majority of injuries in US and UK forces. UK soldiers sustained significantly more of these potentially incapacitating injuries than US troops, 16.1 per 1,000 exits versus 9.1 per 1,000 exits, respectively (chi 2 = 4.07; p = 0.043; relative risk [RR] = 1.70; 95% confidence interval [CI] = 1.01, 2.86). The UK forces sustained significantly more closed head injuries than US forces, 7.1 per 1,000 exits versus 2.3 per 1,000 exits, respectively (chi 2 = 6.4; p = 0.011; RR = 3.13; 95% CI = 1.23, 7.93). The UK forces also had significantly more soldiers with multiple injuries than US forces (RR = 9.15; 95% CI = 2.5, 39.7). Factors that may have influenced differences in injury incidence include differences in weight of personal equipment and possible differences between the drop zones. PMID:9922642

  6. Large exon size does not limit splicing in vivo.

    PubMed

    Chen, I T; Chasin, L A

    1994-03-01

    Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.

  7. 8,9-Dehydrohispanolone-15,16-lactol diterpene prevents LPS-triggered inflammatory responses by inhibiting endothelial activation.

    PubMed

    Jiménez-García, Lidia; Través, Paqui G; López-Fontal, Raquel; Herranz, Sandra; Higueras, María Angeles; de Las Heras, Beatriz; Hortelano, Sonsoles; Luque, Alfonso

    2016-07-15

    Endothelial activation contributes to lung inflammatory disorders by inducing leucocyte recruitment to pulmonary parenchyma. Consequently, vascular-targeted therapies constitute promising strategies for the treatment of inflammatory pathologies. In the present study, we evaluated the effect of 8,9-dehydrohispanolone-15,16-lactol diterpene (DT) on lung endothelium during inflammation. Lung endothelial cells pre-treated with DT and activated with lipopolysaccharide (LPS) or tumour necrosis factor-α (TNF-α) exhibited reduced expression of the pro-inflammatory cytokines Cxcl10, Ccl5 and Cxcl1, whereas the anti-inflammatory molecules IL1r2 and IL-10 were induced. Consistent with this result, DT pre-treatment inhibited nuclear factor κB (NF-κB) nuclear translocation, by interfering with IκBα phosphorylation, and consequently NF-κB transcriptional activity in endothelium activated by LPS or TNF-α. Furthermore, DT, probably through p38 signalling, induced transcriptional activation of genes containing activator protein 1 (AP-1)-binding elements. Inhibition of p38 prevented IL1r2 mRNA expression in endothelium incubated with DT alone or in combination with LPS or TNF-α. Accordingly, conditioned medium (CM) from these cells failed to stimulate leucocytes as measured by a reduction in adhesive ability of the leucocyte cell line J774 to fibronectin (FN). Additionally, DT reduced the expression of the endothelial adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) after activation. Similarly, expression of VCAM-1 and ICAM-1 molecules on the lung endothelial layer of C57/BL6 mice pre-treated with DT and challenged with LPS were unchanged. Finally, inhibition of vascular adhesion molecule expression by DT decreased the interaction of J774 cells with lung endothelial cells in an inflammatory environment. Our findings establish DT as a novel endothelial inhibitor for the treatment of inflammatory

  8. IgA class switch in I alpha exon-deficient mice. Role of germline transcription in class switch recombination.

    PubMed Central

    Harriman, G R; Bradley, A; Das, S; Rogers-Fani, P; Davis, A C

    1996-01-01

    Studies have implicated defective Ig class switch in the pathogenesis of IgA deficiency. To understand better the molecular events that regulate IgA class switch, a 1.4-kb region of the IgA locus containing the I alpha exon was replaced with a human hypoxanthine phosphoribosyltransferase minigene by gene targeting in murine embryonic stem cells. The I alpha exon-deficient mice derived from these embryonic stem cells had normal IgA levels in serum and secretions and normal numbers of IgA B cells in Peyer's patches and spleen. Further, I alpha exon-deficient B cells efficiently underwent IgA class switch in vitro, despite the absence of I alpha exon-containing germline transcripts. Notably, I alpha exon-deficient B cells did not require TGF-beta for IgA class switch since stimulation with LPS alone led to IgA expression. Nonetheless, whereas I alpha exon-deficient B cells constitutively expressed human hypoxanthine phosphoribosyltransferase transcripts, they did not produce IgA in the absence of LPS stimulation. These results demonstrate that the I alpha exon or transcripts containing the I alpha exon are not required for IgA class switch. Further, the effects of TGF-beta on I alpha locus transcription can be supplanted by expression of a heterologous minigene at that locus, but a second signal is required for the induction of IgA class switch. PMID:8567970

  9. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    PubMed Central

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  10. Young People's Participation in Extracurricular Physical Education: A Study of 15-16 Year Olds in North-West England and North-East Wales

    ERIC Educational Resources Information Center

    Smith, Andy; Thurston, Miranda; Green, Ken; Lamb, Kevin

    2007-01-01

    This paper examines the levels and forms of participation in extracurricular physical education (PE) of a cohort of 1010 15-16 year olds attending seven state schools in north-west England and north-east Wales. The data reveal that extracurricular PE provision in all schools retained a particular focus on competitive team sports alongside a number…

  11. Young People's Participation in National Curriculum Physical Education: A Study of 15-16 Year Olds in North-West England and North-East Wales

    ERIC Educational Resources Information Center

    Smith, Andy; Thurston, Miranda; Lamb, Kevin; Green, Ken

    2007-01-01

    Drawing on data from a broader study which investigated the place of sport and physical activity in the lives of 15-16 year olds in England and Wales, this paper examines a relatively neglected dimension of research in physical education, namely, young people's participation in sport and physical activity through National Curriculum Physical…

  12. 47 CFR 90.315 - Special provisions governing use of frequencies in the 476-494 MHz band (TV Channels 15, 16, 17...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... in the 476-494 MHz band (TV Channels 15, 16, 17) in the Southern Louisiana-Texas Offshore Zone. 90..., 17) in the Southern Louisiana-Texas Offshore Zone. (a) The frequency bands from 490-491 and 493-494... Stations by Offshore Stations Operating in the Southern Louisiana-Texas Offshore Zone (65 dB...

  13. 47 CFR 90.315 - Special provisions governing use of frequencies in the 476-494 MHz band (TV Channels 15, 16, 17...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... in the 476-494 MHz band (TV Channels 15, 16, 17) in the Southern Louisiana-Texas Offshore Zone. 90..., 17) in the Southern Louisiana-Texas Offshore Zone. (a) The frequency bands from 490-491 and 493-494... Stations by Offshore Stations Operating in the Southern Louisiana-Texas Offshore Zone (65 dB...

  14. 47 CFR 90.315 - Special provisions governing use of frequencies in the 476-494 MHz band (TV Channels 15, 16, 17...

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... in the 476-494 MHz band (TV Channels 15, 16, 17) in the Southern Louisiana-Texas Offshore Zone. 90..., 17) in the Southern Louisiana-Texas Offshore Zone. (a) The frequency bands from 490-491 and 493-494... Stations by Offshore Stations Operating in the Southern Louisiana-Texas Offshore Zone (65 dB...

  15. 47 CFR 90.315 - Special provisions governing use of frequencies in the 476-494 MHz band (TV Channels 15, 16, 17...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... in the 476-494 MHz band (TV Channels 15, 16, 17) in the Southern Louisiana-Texas Offshore Zone. 90..., 17) in the Southern Louisiana-Texas Offshore Zone. (a) The frequency bands from 490-491 and 493-494... Stations by Offshore Stations Operating in the Southern Louisiana-Texas Offshore Zone (65 dB...

  16. Triple-layer dissection of the lung adenocarcinoma transcriptome: regulation at the gene, transcript, and exon levels.

    PubMed

    Hsu, Min-Kung; Wu, I-Ching; Cheng, Ching-Chia; Su, Jen-Liang; Hsieh, Chang-Huain; Lin, Yeong-Shin; Chen, Feng-Chi

    2015-10-01

    Lung adenocarcinoma is one of the most deadly human diseases. However, the molecular mechanisms underlying this disease, particularly RNA splicing, have remained underexplored. Here, we report a triple-level (gene-, transcript-, and exon-level) analysis of lung adenocarcinoma transcriptomes from 77 paired tumor and normal tissues, as well as an analysis pipeline to overcome genetic variability for accurate differentiation between tumor and normal tissues. We report three major results. First, more than 5,000 differentially expressed transcripts/exonic regions occur repeatedly in lung adenocarcinoma patients. These transcripts/exonic regions are enriched in nicotine metabolism and ribosomal functions in addition to the pathways enriched for differentially expressed genes (cell cycle, extracellular matrix receptor interaction, and axon guidance). Second, classification models based on rationally selected transcripts or exonic regions can reach accuracies of 0.93 to 1.00 in differentiating tumor from normal tissues. Of the 28 selected exonic regions, 26 regions correspond to alternative exons located in such regulators as tumor suppressor (GDF10), signal receptor (LYVE1), vascular-specific regulator (RASIP1), ubiquitination mediator (RNF5), and transcriptional repressor (TRIM27). Third, classification systems based on 13 to 14 differentially expressed genes yield accuracies near 100%. Genes selected by both detection methods include C16orf59, DAP3, ETV4, GABARAPL1, PPAR, RADIL, RSPO1, SERTM1, SRPK1, ST6GALNAC6, and TNXB. Our findings imply a multilayered lung adenocarcinoma regulome in which transcript-/exon-level regulation may be dissociated from gene-level regulation. Our described method may be used to identify potentially important genes/transcripts/exonic regions for the tumorigenesis of lung adenocarcinoma and to construct accurate tumor vs. normal classification systems for this disease.

  17. Delineation of the Marfan phenotype associated with mutations in exons 23-32 of the FBN1 gene

    SciTech Connect

    Putnam, E.A.; Cho, M.; Milewicz, D.M.

    1996-03-29

    Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-based exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 mutations in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24-32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling. 49 refs., 4 figs., 2 tabs.

  18. The second exon-encoded factor XII region is involved in the interaction of factor XII with factor XI and does not contribute to the binding site for negatively charged surfaces.

    PubMed

    Citarella, F; Fedele, G; Roem, D; Fantoni, A; Hack, C E

    1998-12-01

    Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-triangle up19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-triangle up19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-triangle up19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-triangle up19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-triangle up19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-triangle up19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-triangle up19 bound to kaolin, which binding was equally well inhibited by either, rFXII-triangle up19 or full-length FXII (IC50 = 7.2 +/- 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-triangle up19 generated a similar amount of FXIIa- and kallikrein-C1-inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor

  19. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy.

    PubMed Central

    Nguyen, T M; Morris, G E

    1993-01-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. Images Figure 4 Figure 1 PMID:7684887

  20. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    SciTech Connect

    Nguyen thi Man; Morris, G.E. )

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  1. Exon organization of the human FKBP-12 gene: Correlation with structural and functional protein domains

    SciTech Connect

    DiLella, A.G.; Craig, R.J. )

    1991-09-03

    FKBP-12, the major T-cell binding protein for the immunosuppressive agents FK506 and rapamycin, catalyzes the interconversion of the cis and trans rotamers of the peptidyl-prolyl amide bond of peptide and protein substrates. The function of rotamase activity in cells and the role of FKBP-12 in immunoregulation is uncertain. In this paper the authors report the cloning and characterization of the human chromosomal FKBP-12 gene and four processed FKBP-12 pseudogenes. The FKBP-12 gene is 24 kilobases in length and contains five exons. The protein-coding region of the gene is divided into four exon modules that correlate with the structural and functional domains of the protein. The novel structure of FKBP-12 resulting form the topology of the antiparallel {beta}-sheet is the topological crossing of two loops that are encoded by separate exons. Separate exons also encode the antiparallel {beta}-sheet and {alpha}-helical region that define the drug-binding pocket and enzyme activity site of FKBP-12. The exon organization of the FKBP-12 gene structure will enable inactivation of this gene by homologous recombination in cells to provide a model to study the role of FKBP-12 in immunoregulation and normal cellular processes.

  2. GeneAlign: a coding exon prediction tool based on phylogenetical comparisons.

    PubMed

    Hsieh, Shu Ju; Lin, Chun Yuan; Liu, Ning Han; Chow, Wei Yuan; Tang, Chuan Yi

    2006-07-01

    GeneAlign is a coding exon prediction tool for predicting protein coding genes by measuring the homologies between a sequence of a genome and related sequences, which have been annotated, of other genomes. Identifying protein coding genes is one of most important tasks in newly sequenced genomes. With increasing numbers of gene annotations verified by experiments, it is feasible to identify genes in the newly sequenced genomes by comparing to annotated genes of phylogenetically close organisms. GeneAlign applies CORAL, a heuristic linear time alignment tool, to determine if regions flanked by the candidate signals (initiation codon-GT, AG-GT and AG-STOP codon) are similar to annotated coding exons. Employing the conservation of gene structures and sequence homologies between protein coding regions increases the prediction accuracy. GeneAlign was tested on Projector dataset of 491 human-mouse homologous sequence pairs. At the gene level, both the average sensitivity and the average specificity of GeneAlign are 81%, and they are larger than 96% at the exon level. The rates of missing exons and wrong exons are smaller than 1%. GeneAlign is a free tool available at http://genealign.hccvs.hc.edu.tw.

  3. Tissue-specific expression of the bovine aromatase-encoding gene uses multiple transcriptional start sites and alternative first exons.

    PubMed

    Fürbass, R; Kalbe, C; Vanselow, J

    1997-07-01

    Here we report on the genomic structure of the bovine aromatase cytochrome P450-encoding gene (Cyp19) and its tissue-specific transcript variants. The gene comprises at least 14 exons (1.1, 1.2a, 1.2b, 1.3,1.4, and 2-10) spanning more than 56 kilobases of genomic DNA. The coding area is confined to exons 2-10. Transcriptional start sites of Cyp19 were examined in granulosa cells, placenta, testis, adrenal gland, and brain, employing 5'-RACE (rapid amplification of complementary DNA ends) and primer extension. The analysis of 5'-RACE clones revealed six Cyp19 transcript variants that were different within their 5'-untranslated regions (5'-UTR). Yet, the coding region was identical in all clones. Although two of these 5'-UTR (the first 152 nucleotides of exon 2 and exon 1.4) are conserved among different species, four others (exons 1.1, 1.2a, 1.2b, and 1.3) did not show sequence homology to any other species. Transcription from exons 1.1 and 2 starts at several adjacent sites. In granulosa cells and placenta, but not in brain, a fraction of transcripts starting with exon 1.2a contains an additional untranslated exon, 1.2b, due to alternative splicing. Transcript variants comprising exon 1.1, 1.2a, 1.2b, or 1.3 were mainly found in the placenta, those with the 5'-UTR of exon 2 were predominant in granulosa cells, and transcripts with exon 1.4 prevailed in the brain. Estimates of Cyp19 transcript concentrations in six different tissues revealed high levels in granulosa cells and placenta, intermediate levels in testis and brain, and low levels in adrenal gland and liver. Our experiments demonstrate that six transcript variants of the bovine Cyp19 gene, including 9-11 exons, are expressed with tissue-specific preferences. These transcripts are presumably generated using five different promoter regions and tissue-specific alternative splicing. PMID:9202222

  4. Vega landing sites - Venera 15/16 unit analogs from Pioneer Venus reflectivity and RMS slope data

    NASA Technical Reports Server (NTRS)

    Bindschadler, Duane L.; Head, James W., III; Garvin, James B.

    1986-01-01

    Pioneer Venus radar data on surface properties have been used to compare the Vega spacecraft landing sites with the northern 1/4 of Venus mapped by the orbiters Venera 15 and 16. The regions surrounding both landing sites possess surface reflectivity and small-scale roughness properties most similar to those of mapped volcanoes and volcanic plains regions and different surface properties than those of mapped tectonic units. Regions analogous to the Vega 1 site are relatively rare, covering 2.8 percent of the mapped surface. Vega 2 analogs are much more common and cover 22.6 percent of the surface. Neither landing site is representative of the nearby highlands of Aphrodite, but the Vega 2 landing site is similar to much of the northern plains of Venus.

  5. CPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte Maturation

    PubMed Central

    Wilczynska, Ania; Git, Anna; Argasinska, Joanna; Belloc, Eulàlia; Standart, Nancy

    2016-01-01

    Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3’UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3’UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways. PMID:26829217

  6. Foldons, Protein Structural Modules, and Exons

    NASA Astrophysics Data System (ADS)

    Panchenko, Anna R.; Luthey-Schulten, Zaida; Wolynes, Peter G.

    1996-03-01

    Foldons, which are kinetically competent, quasi-independently folding units of a protein, may be defined using energy landscape analysis. Foldons can be identified by maxima in a scan of the ratio of a contiguous segment's energetic stability gap to the energy variance of that segment's molten globule states, reflecting the requirement of minimal frustration. The predicted foldons are compared with the exons and structural modules for 16 of the 30 proteins studied. Statistical analysis indicates a strong correlation between the energetically determined foldons and Go's geometrically defined structural modules, but there are marked sequence-dependent effects. There is only a weak correlation of foldons to exons. For γ II-crystallin, myoglobin, barnase, α -lactalbumin, and cytochrome c the foldons and some noncontiguous clusters of foldons compare well with intermediates observed in experiment.

  7. 78 FR 51206 - Notice of October 15-16, 2013, Meeting of the National Park System Advisory Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-20

    ... be briefed on sites within the National Capital Region. The Board will convene its business meeting... National Park Service officials regarding education, leadership development and science; deliberate and...) Program NHL Program matters will be considered at the morning session of the business meeting on...

  8. A majority of m6A residues are in the last exons, allowing the potential for 3' UTR regulation.

    PubMed

    Ke, Shengdong; Alemu, Endalkachew A; Mertens, Claudia; Gantman, Emily Conn; Fak, John J; Mele, Aldo; Haripal, Bhagwattie; Zucker-Scharff, Ilana; Moore, Michael J; Park, Christopher Y; Vågbø, Cathrine Broberg; Kusśnierczyk, Anna; Klungland, Arne; Darnell, James E; Darnell, Robert B

    2015-10-01

    We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice. PMID:26404942

  9. A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation

    PubMed Central

    Ke, Shengdong; Alemu, Endalkachew A.; Mertens, Claudia; Gantman, Emily Conn; Fak, John J.; Mele, Aldo; Haripal, Bhagwattie; Zucker-Scharff, Ilana; Moore, Michael J.; Park, Christopher Y.; Vågbø, Cathrine Broberg; Kusśnierczyk, Anna; Klungland, Arne; Darnell, James E.; Darnell, Robert B.

    2015-01-01

    We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m6A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3′-most (last) exons, with a very sharp rise (sixfold) within 150–400 nucleotides of the start of the last exon. Two-thirds of last exon m6A and >40% of all m6A in mRNA are present in 3′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m6A sites around stop codons. Moreover, m6A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m6A density peaks early in the 3′ UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m6A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m6A modification in regulating proximal alternative polyA choice. PMID:26404942

  10. Whole-Exome Enrichment with the Agilent SureSelect Human All Exon Platform.

    PubMed

    Chen, Rui; Im, Hogune; Snyder, Michael

    2015-07-01

    There are multiple platforms available for whole-exome enrichment and sequencing (WES). This protocol is based on the Agilent SureSelect Human All Exon platform, which targets ∼50 Mb of the human exonic regions. The SureSelect system uses ∼120-base RNA probes to capture known coding DNA sequences (CDS) from the NCBI Consensus CDS Database as well as other major RNA coding sequence databases, such as Sanger miRBase. The protocol can be performed at the benchside without the need for automation, and the resulting library can be used for targeted next-generation sequencing on an Illumina HiSeq 2000 sequencer.

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

    SciTech Connect

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

    2008-11-07

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

  12. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

    PubMed Central

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a “splicing memory” hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

  13. Statistical analysis of the exon-intron structure of higher and lower eukaryote genes.

    PubMed

    Kriventseva, E V; Gelfand, M S

    1999-10-01

    Statistics of the exon-intron structure and splicing sites of several diverse eukaryotes was studied. The yeast exon-intron structures have a number of unique features. A yeast gene usually have at most one intron. The branch site is strongly conserved, whereas the polypirimidine tract is short. Long yeast introns tend to have stronger acceptor sites. In other species the branch site is less conserved and often cannot be determined. In non-yeast samples there is an almost universal correlation between lengths of neighboring exons (all samples excluding protists) and correlation between lengths of neighboring introns (human, drosophila, protists). On the average first introns are longer, and anomalously long introns are usually first introns in a gene. There is a universal preference for exons and exon pairs with the (total) length divisible by 3. Introns positioned between codons are preferred, whereas those positioned between the first and second positions in codon are avoided. The choice of A or G at the third position of intron (the donor splice sites generally prefer purines at this position) is correlated with the overall GC-composition of the gene. In all samples dinucleotide AG is avoided in the region preceding the acceptor site.

  14. Inappropriate splicing of a chimeric gene containing a large internal exon results in exon skipping in transgenic mice.

    PubMed

    Davisson, R L; Nuutinen, N; Coleman, S T; Sigmund, C D

    1996-10-15

    We generated transgenic mice containing a chimeric construct consisting of the alpha-cardiac myosin heavy chain (alpha cMHC) promoter and the human renin (hRen) gene in order to target hRen synthesis specifically to the heart. The construct consisted of three segments: (i) an alpha cMHC DNA segment including 4.5 kb of 5' flanking DNA and an additional 1.1 kb of genomic DNA encompassing exons I-III (non-coding) and the first two introns; (ii) a partial hRen cDNA consisting of exons I-VI; and (iii) a hRen genomic segment containing exons VII through IX, their intervening introns, and 400 bp of 3' flanking DNA. This results in the formation of a 909 bp internal fusion exon consisting of alpha cMHC, polylinker, and hRen sequences. Despite the presence of splice acceptor and donor sites bracketing this exon, transcription of this transgene resulted in a major alternatively spliced mRNA lacking the exon and therefore a majority of the hRen coding sequence. Cloning and sequencing of RT-PCR products from several heart samples from two independent transgenic lines confirmed accurate and faithful splicing of alpha cMHC exon II to hRen exon VII thus bypassing the internal fusion exon. All other exons (alpha cMHC exons I and II and hRen exons VII, VIII and IX) were appropriately spliced. These results are consistent with the hypothesis on exon definition which states that internal exons have a size limitation. Moreover, the results demonstrate that transgenes present in the genome at independent insertion sites and in either a single copy or multiple copies can be subject to exon skipping. The implications for transgene design will be discussed.

  15. Exon capture optimization in amphibians with large genomes.

    PubMed

    McCartney-Melstad, Evan; Mount, Genevieve G; Shaffer, H Bradley

    2016-09-01

    Gathering genomic-scale data efficiently is challenging for nonmodel species with large, complex genomes. Transcriptome sequencing is accessible for organisms with large genomes, and sequence capture probes can be designed from such mRNA sequences to enrich and sequence exonic regions. Maximizing enrichment efficiency is important to reduce sequencing costs, but relatively few data exist for exon capture experiments in nonmodel organisms with large genomes. Here, we conducted a replicated factorial experiment to explore the effects of several modifications to standard protocols that might increase sequence capture efficiency for amphibians and other taxa with large, complex genomes. Increasing the amounts of c0 t-1 repetitive sequence blocker and individual input DNA used in target enrichment reactions reduced the rates of PCR duplication. This reduction led to an increase in the percentage of unique reads mapping to target sequences, essentially doubling overall efficiency of the target capture from 10.4% to nearly 19.9% and rendering target capture experiments more efficient and affordable. Our results indicate that target capture protocols can be modified to efficiently screen vertebrates with large genomes, including amphibians. PMID:27223337

  16. Exon duplications in the ATP7A gene: Frequency and Transcriptional Behaviour

    PubMed Central

    2011-01-01

    Background Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene. Methods The ATP7A gene was screened for exon duplications using multiplex ligation-dependent probe amplification (MLPA). The expression level of ATP7A was investigated by real-time PCR and detailed analysis of the ATP7A mRNA was performed by RT-PCR followed by sequencing. In order to investigate whether the identified duplicated fragments originated from a single or from two different X-chromosomes, polymorphic markers located in the duplicated fragments were analyzed. Results Partial ATP7A gene duplication was identified in 20 unrelated patients including one patient with Occipital Horn Syndrome (OHS). Duplications in the ATP7A gene are estimated from our material to be the disease causing mutation in 4% of the Menkes disease patients. The duplicated regions consist of between 2 and 15 exons. In at least one of the cases, the duplication was due to an intra-chromosomal event. Characterization of the ATP7A mRNA transcripts in 11 patients revealed that the duplications were organized in tandem, in a head to tail direction. The reading frame was disrupted in all 11 cases. Small amounts of wild-type transcript were found in all patients as a result of exon-skipping events occurring in the duplicated regions. In the OHS patient with a duplication of exon 3 and 4, the duplicated out-of-frame transcript coexists with an almost equally represented wild-type transcript, presumably leading to the milder phenotype. Conclusions In general, patients with duplication of only 2 exons exhibit a milder phenotype as compared to patients with duplication of more than 2 exons. This study provides insight into exon duplications in the ATP7A gene. PMID:22074552

  17. Detection EGFR exon 19 status of lung cancer patients by DNA electrochemical biosensor.

    PubMed

    Xu, Xiong-Wei; Weng, Xiu-Hua; Wang, Chang-Lian; Lin, Wei-Wei; Liu, Ai-Lin; Chen, Wei; Lin, Xin-Hua

    2016-06-15

    Epidermal growth factor receptor (EGFR) exon 19 mutation status is a very important prediction index for tyrosine kinase inhibitors (TKIs) therapy. In this paper, we constructed a superior selective sandwich-type electrochemical biosensor to detect in-frame deletions in exon 19 of EGFR in real samples of patients with non-small cell lung carcinoma. Based on the characteristics of different hybridization efficiency in different hybridization phase conditions, different region around EGFR exon 19 deletion hotspots was selected to design DNA probes to improve biosensor performance. The results confirm that alteration of deletion location in target deliberately according to different hybridization phase is able to improve selectivity of sandwich-type DNA biosensor. Satisfactory discrimination ability can be achieved when the deletions are located in the capture probe interaction region. In order to improve efficiency of ssDNA generation from dsDNA, we introduce Lambda exonuclease (λ-exo) to sandwich-type biosensor system. EGFR exon 19 statuses of clinical real samples from lung cancer patients can be discriminated successfully by the proposed method. Our research would make the electrochemical biosensor be an excellent candidate for EGFR detection for lung cancer patients. PMID:26874108

  18. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3

    PubMed Central

    Toonen, Lodewijk J. A.; Schmidt, Iris; Luijsterburg, Martijn S.; van Attikum, Haico; van Roon-Mom, Willeke M. C.

    2016-01-01

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3. PMID:27731380

  19. Cooperative binding of TIA-1 and U1 snRNP in K-SAM exon splicing activation

    SciTech Connect

    Gesnel, Marie-Claude; Theoleyre, Sandrine; Del Gatto-Konczak, Fabienne; Breathnach, Richard . E-mail: breathna@nantes.inserm.fr

    2007-07-13

    In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.

  20. An evaluation of transcriptome-based exon capture for frog phylogenomics across multiple scales of divergence (Class: Amphibia, Order: Anura).

    PubMed

    Portik, Daniel M; Smith, Lydia L; Bi, Ke

    2016-09-01

    Custom sequence capture experiments are becoming an efficient approach for gathering large sets of orthologous markers in nonmodel organisms. Transcriptome-based exon capture utilizes transcript sequences to design capture probes, typically using a reference genome to identify intron-exon boundaries to exclude shorter exons (<200 bp). Here, we test directly using transcript sequences for probe design, which are often composed of multiple exons of varying lengths. Using 1260 orthologous transcripts, we conducted sequence captures across multiple phylogenetic scales for frogs, including outgroups ~100 Myr divergent from the ingroup. We recovered a large phylogenomic data set consisting of sequence alignments for 1047 of the 1260 transcriptome-based loci (~561 000 bp) and a large quantity of highly variable regions flanking the exons in transcripts (~70 000 bp), the latter improving substantially by only including ingroup species (~797 000 bp). We recovered both shorter (<100 bp) and longer exons (>200 bp), with no major reduction in coverage towards the ends of exons. We observed significant differences in the performance of blocking oligos for target enrichment and nontarget depletion during captures, and differences in PCR duplication rates resulting from the number of individuals pooled for capture reactions. We explicitly tested the effects of phylogenetic distance on capture sensitivity, specificity, and missing data, and provide a baseline estimate of expectations for these metrics based on a priori knowledge of nuclear pairwise differences among samples. We provide recommendations for transcriptome-based exon capture design based on our results, cost estimates and offer multiple pipelines for data assembly and analysis. PMID:27241806

  1. An evaluation of transcriptome-based exon capture for frog phylogenomics across multiple scales of divergence (Class: Amphibia, Order: Anura).

    PubMed

    Portik, Daniel M; Smith, Lydia L; Bi, Ke

    2016-09-01

    Custom sequence capture experiments are becoming an efficient approach for gathering large sets of orthologous markers in nonmodel organisms. Transcriptome-based exon capture utilizes transcript sequences to design capture probes, typically using a reference genome to identify intron-exon boundaries to exclude shorter exons (<200 bp). Here, we test directly using transcript sequences for probe design, which are often composed of multiple exons of varying lengths. Using 1260 orthologous transcripts, we conducted sequence captures across multiple phylogenetic scales for frogs, including outgroups ~100 Myr divergent from the ingroup. We recovered a large phylogenomic data set consisting of sequence alignments for 1047 of the 1260 transcriptome-based loci (~561 000 bp) and a large quantity of highly variable regions flanking the exons in transcripts (~70 000 bp), the latter improving substantially by only including ingroup species (~797 000 bp). We recovered both shorter (<100 bp) and longer exons (>200 bp), with no major reduction in coverage towards the ends of exons. We observed significant differences in the performance of blocking oligos for target enrichment and nontarget depletion during captures, and differences in PCR duplication rates resulting from the number of individuals pooled for capture reactions. We explicitly tested the effects of phylogenetic distance on capture sensitivity, specificity, and missing data, and provide a baseline estimate of expectations for these metrics based on a priori knowledge of nuclear pairwise differences among samples. We provide recommendations for transcriptome-based exon capture design based on our results, cost estimates and offer multiple pipelines for data assembly and analysis.

  2. Exon-intron structure of the human neuronal nicotinic acetylcholine receptor {alpha}4 subunit (CHRNA4)

    SciTech Connect

    Steinlein, O.; Weiland, S.; Stoodt, J.; Propping, P.

    1996-03-01

    The human neuronal nicotinic acetylcholine receptor {alpha}4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set of primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idopathic epilepsies. 29 refs., 3 figs., 1 tab.

  3. Exon redefinition by a point mutation within exon 5 of the glucose-6-phosphatase gene is the major cause of glycogen storage disease type 1a in Japan

    SciTech Connect

    Kajihara, Susumu; Yamamoto, Kyosuke; Kido, Keiko

    1995-09-01

    Glycogen storage disease (GSD) type 1a (von Gierke disease) is an autosomal recessive disorder caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). We have identified a novel mutation in the G6Pase gene of a individual with GSD type 1a. The cDNA from the patient`s liver revealed a 91-nt deletion in exon 5. The genomic DNA from the patient`s white blood cells revealed no deletion or mutation at the splicing junction of intron 4 and exon 5. The 3{prime} splicing occurred 91 bp from the 5{prime} site of exon 5 (at position 732 in the coding region), causing a substitution of a single nucleotide (G to T) at position 727 in the coding region. Further confirmation of the missplicing was obtained by transient expression of allelic minigene constructs into animal cells. Another eight unrelated families of nine Japanese patients were all found to have this mutation. This mutation is a new type of splicing mutation in the G6Pase gene, and 91% of patients and carriers suffering from GSD1a in Japan are detectable with this splicing mutation. 28 refs., 5 figs., 2 tabs.

  4. Use of fake identification to purchase alcohol amongst 15-16 year olds: a cross-sectional survey examining alcohol access, consumption and harm

    PubMed Central

    2010-01-01

    Background Despite legislation and enforcement activities to prevent underage access to alcohol, underage individuals continue to be able to access alcohol and to do so at levels which put them at significant risk of alcohol-related harm. Methods An opportunistic survey of 15-16 year olds (n = 9,833) across North West England was used to examine alcohol consumption, methods of access and related harms experienced (such as regretted sex). Associations between these were analysed using chi square and logistic regression techniques. Results Over a quarter (28.3%) of 15-16 year old participants who drank reported having bought their own alcohol. One seventh (14.9%) of these owned at least one form of fake identification for which by far the most common purchase method was online. Logistic regression analyses showed that those who owned fake identification were significantly more likely to be male (AOR = 2.0; 95% CI = 1.7-2.5; P < 0.001) and to receive a higher personal weekly income (comparing those who received > £30 with those who received ≤ £10: AOR = 3.7; 95% CI = 2.9-4.9; P < 0.001). After taking into account differences in demographic characteristics and personal weekly income, ownership of fake identification was significantly associated with binge drinking (AOR = 3.5, 95% CI = 2.8-4.3; P < 0.001), frequent drinking (AOR = 3.0, 95% CI = 2.5-3.7; P < 0.001) and public drinking (AOR = 3.3, 95% CI = 2.5-4.1; P < 0.001) compared with those who did not own fake identification. Further, those who reported owning fake identification were significantly more likely to report experiencing a variety of alcohol-related harms such as regretted sex after drinking (chi square, all P < 0.001). Conclusions Young people (aged 15-16 years) who have access to fake identification are at a particularly high risk of reporting hazardous alcohol consumption patterns and related harm. Owning fake identification should be considered a risk factor for involvement in risky drinking

  5. SNP discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate.

    PubMed

    Roffler, Gretchen H; Amish, Stephen J; Smith, Seth; Cosart, Ted; Kardos, Marty; Schwartz, Michael K; Luikart, Gordon

    2016-09-01

    Identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. We used targeted resequencing to identify SNP markers in 5321 candidate adaptive genes associated with known immunological, metabolic and growth functions in ovids and other ungulates. We selectively targeted 8161 exons in protein-coding and nearby 5' and 3' untranslated regions of chosen candidate genes. Targeted sequences were taken from bighorn sheep (Ovis canadensis) exon capture data and directly from the domestic sheep genome (Ovis aries v. 3; oviAri3). The bighorn sheep sequences used in the Dall's sheep (Ovis dalli dalli) exon capture aligned to 2350 genes on the oviAri3 genome with an average of 2 exons each. We developed a microfluidic qPCR-based SNP chip to genotype 476 Dall's sheep from locations across their range and test for patterns of selection. Using multiple corroborating approaches (lositan and bayescan), we detected 28 SNP loci potentially under selection. We additionally identified candidate loci significantly associated with latitude, longitude, precipitation and temperature, suggesting local environmental adaptation. The three methods demonstrated consistent support for natural selection on nine genes with immune and disease-regulating functions (e.g. Ovar-DRA, APC, BATF2, MAGEB18), cell regulation signalling pathways (e.g. KRIT1, PI3K, ORRC3), and respiratory health (CYSLTR1). Characterizing adaptive allele distributions from novel genetic techniques will facilitate investigation of the influence of environmental variation on local adaptation of a northern alpine ungulate throughout its range. This research demonstrated the utility of exon capture for gene-targeted SNP discovery and subsequent SNP chip genotyping using low-quality samples in a nonmodel species. PMID:27327375

  6. SNP discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate.

    PubMed

    Roffler, Gretchen H; Amish, Stephen J; Smith, Seth; Cosart, Ted; Kardos, Marty; Schwartz, Michael K; Luikart, Gordon

    2016-09-01

    Identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. We used targeted resequencing to identify SNP markers in 5321 candidate adaptive genes associated with known immunological, metabolic and growth functions in ovids and other ungulates. We selectively targeted 8161 exons in protein-coding and nearby 5' and 3' untranslated regions of chosen candidate genes. Targeted sequences were taken from bighorn sheep (Ovis canadensis) exon capture data and directly from the domestic sheep genome (Ovis aries v. 3; oviAri3). The bighorn sheep sequences used in the Dall's sheep (Ovis dalli dalli) exon capture aligned to 2350 genes on the oviAri3 genome with an average of 2 exons each. We developed a microfluidic qPCR-based SNP chip to genotype 476 Dall's sheep from locations across their range and test for patterns of selection. Using multiple corroborating approaches (lositan and bayescan), we detected 28 SNP loci potentially under selection. We additionally identified candidate loci significantly associated with latitude, longitude, precipitation and temperature, suggesting local environmental adaptation. The three methods demonstrated consistent support for natural selection on nine genes with immune and disease-regulating functions (e.g. Ovar-DRA, APC, BATF2, MAGEB18), cell regulation signalling pathways (e.g. KRIT1, PI3K, ORRC3), and respiratory health (CYSLTR1). Characterizing adaptive allele distributions from novel genetic techniques will facilitate investigation of the influence of environmental variation on local adaptation of a northern alpine ungulate throughout its range. This research demonstrated the utility of exon capture for gene-targeted SNP discovery and subsequent SNP chip genotyping using low-quality samples in a nonmodel species.

  7. Characterization of Major Histocompatibility Complex (MHC) DRB Exon 2 and DRA Exon 3 Fragments in a Primary Terrestrial Rabies Vector (Procyon lotor)

    PubMed Central

    Castillo, Sarrah; Srithayakumar, Vythegi; Meunier, Vanessa; Kyle, Christopher J.

    2010-01-01

    The major histocompatibility complex (MHC) presents a unique system to explore links between genetic diversity and pathogens, as diversity within MHC is maintained in part by pathogen driven selection. While the majority of wildlife MHC studies have investigated species that are of conservation concern, here we characterize MHC variation in a common and broadly distributed species, the North American raccoon (Procyon lotor). Raccoons host an array of broadly distributed wildlife diseases (e.g., canine distemper, parvovirus and raccoon rabies virus) and present important human health risks as they persist in high densities and in close proximity to humans and livestock. To further explore how genetic variation influences the spread and maintenance of disease in raccoons we characterized a fragment of MHC class II DRA exon 3 (250bp) and DRB exon 2 (228 bp). MHC DRA was found to be functionally monomorphic in the 32 individuals screened; whereas DRB exon 2 revealed 66 unique alleles among the 246 individuals screened. Between two and four alleles were observed in each individual suggesting we were amplifying a duplicated DRB locus. Nucleotide differences between DRB alleles ranged from 1 to 36 bp (0.4–15.8% divergence) and translated into 1 to 21 (1.3–27.6% divergence) amino acid differences. We detected a significant excess of nonsynonymous substitutions at the peptide binding region (P = 0.005), indicating that DRB exon 2 in raccoons has been influenced by positive selection. These data will form the basis of continued analyses into the spatial and temporal relationship of the raccoon rabies virus and the immunogenetic response in its primary host. PMID:20706587

  8. MIR retroposon exonization promotes evolutionary variability and generates species-specific expression of IGF-1 splice variants.

    PubMed

    Annibalini, Giosuè; Bielli, Pamela; De Santi, Mauro; Agostini, Deborah; Guescini, Michele; Sisti, Davide; Contarelli, Serena; Brandi, Giorgio; Villarini, Anna; Stocchi, Vilberto; Sette, Claudio; Barbieri, Elena

    2016-05-01

    Insulin-like growth factor (IGF) -1 is a pleiotropic hormone exerting mitogenic and anti-apoptotic effects. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. The biological significance of the IGF-1 splice variants and how the E-peptides affect the actions of mature IGF-1 are largely unknown. In this study we investigated the origin and conservation of the IGF-1 E-peptides and we compared the pattern of expression of the IGF-1 isoforms in vivo, in nine mammalian species, and in vitro using human and mouse IGF-1 minigenes. Our analysis showed that only IGF-1Ea is conserved among all vertebrates, whereas IGF-1Eb and IGF-1Ec are an evolutionary novelty originated from the exonization of a mammalian interspersed repetitive-b (MIR-b) element. Both IGF-1Eb and IGF-1Ec mRNAs were constitutively expressed in all mammalian species analyzed but their expression ratio varies greatly among species. Using IGF-1 minigenes we demonstrated that divergence in cis-acting regulatory elements between human and mouse conferred species-specific features to the exon 5 region. Finally, the protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains. In conclusion, exonization of a MIR-b element in the IGF-1 gene determined gain of exon 5 during mammalian evolution. Alternative splicing of this novel exon added new regulatory elements at the mRNA and protein level potentially able to regulate the mature IGF-1 across tissues and species. PMID:27048986

  9. Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales

    PubMed Central

    2012-01-01

    Background To date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias) based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species. Results We selectively targeted 11,975 exons (~4 Mb) on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1%) across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My), however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified. Conclusions Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life. PMID:22900609

  10. Exons 16 and 17 of the amyloid precursor protein gene in familial inclusion body myopathy.

    PubMed

    Sivakumar, K; Cervenáková, L; Dalakas, M C; Leon-Monzon, M; Isaacson, S H; Nagle, J W; Vasconcelos, O; Goldfarb, L G

    1995-08-01

    Accumulation of beta-amyloid protein (A beta) occurs in some muscle fibers of patients with inclusion body myopathy and resembles the type of amyloid deposits seen in the affected tissues of patients with Alzheimer's disease and cerebrovascular amyloidosis. Because mutations in exons 16 and 17 of the beta-amyloid precursor protein (beta APP) gene on chromosome 21 have been identified in patients with early-onset familial Alzheimer's disease and Dutch-type cerebrovascular amyloidosis, we searched for mutations of the same region in patients with familial inclusion body myopathy. Sequencing of both alleles in 8 patients from four unrelated families did not reveal any mutations in these exons. The amyloid deposition in familial forms of inclusion body myopathy may be either due to errors in other gene loci, or it is secondary reflecting altered beta APP metabolism or myocyte degeneration and cell membrane degradation.

  11. 15/16 ips Operation of the Precision Instrument Company Model P15100 tape recorder to record the standard (30 Hz) NCER seismic data multiplex system

    USGS Publications Warehouse

    Eaton, Jerry P.

    1976-01-01

    In recent months the need has arisen to record special seismic networks consisting of a dozen or more standard NCER seismic systems telemetered to a central collection point on a reliable, portable, low-power tape recorder. Because of its simplicity and the ease with which it can be adapted for the purpose, the PI 5100 field recorder should be considered for such use. In the tests described here, a PI 5100 was speeded up to run at 15/16 inches per second (ips) and signals from the standard multiplex system test modulator bank were recorded on one tape track by means of a simple, improvised AM record amplifier. The results of these tests are extremely encouraging: the dynamic range of the system when played back on the Bell and Howell Model 3700 B reproduce machine, with subtractive compensation, is nearly as high as for the system employing the B&H 3700 B for recording. These notes indicate the principle employed to speed up the recorder, outline the circuit required to drive the tape heads in the AM record mode, and describe the tests carried out to evaluate the system's performance.

  12. What are the barriers which discourage 15-16 year-old girls from participating in team sports and how can we overcome them?

    PubMed

    Wetton, Abigail R; Radley, Rebecca; Jones, Angela R; Pearce, Mark S

    2013-01-01

    Given the clear benefits of regular physical activity (such as reduced risks of cardiovascular disease and obesity, as well as other benefits including those related to mental health), exploration of the reasons that adolescent girls give for not taking part in team sports may be particularly valuable for enhancing later rates of participation. We combined questionnaires (n = 60) and semistructured interviews (n = 6) to assess the barriers that prevent 15-16-year-old girls from participating in extracurricular team games and what can be done to overcome these barriers and improve physical activity levels. Four barriers became prominent as to why girls in this sample do not participate: Internal Factors, Existing Stereotypes, Other Hobbies and Teachers. Methods to overcome these barriers were identified; changing teachers' attitudes and shifting the media's focus away from male sport. Following the successful summer Olympics and Paralympics in the UK, and the resulting positive focus on some of the nation's female athletes, a shift in focus may be possible. However, this needs to be maintained to allow girls more opportunities, role models and motivation to participate in sport. PMID:24073416

  13. Eu3+/Sm3+ hybrids based with 8-hydroxybenz[de]anthracen-7-one organically modified mesoporous silica SBA-15/16

    NASA Astrophysics Data System (ADS)

    Gu, Yan-Jing; Yan, Bing

    2015-12-01

    A series of organic-inorganic hybrid materials were prepared by linking lanthanide (Eu3+, Sm3+) complexes to mesoporous SBA-15/SBA-16 through 8-hydroxybenz[de]anthracen-7-one modified silane (HBA-Si) as linker. The physical characterizations of these hybrids revealed that they all have high surface area, uniformity in mesostructure. The luminescence properties of these covalently bonded materials (denoted as Ln(HBA-SBA-15)3phen and Ln(HBA-SBA-16)3phen) were compared with ternary complexes (Ln(HBA)3phen) (Ln = Eu, Sm). Eu(HBA-SBA-15(16))3phen hybrids display better thermal stability, whose luminescent lifetimes and quantum efficiencies were matchable with those of Eu(HBA)3phen complex in spite of its much lower effective condensation of Eu3+ species. In addition, the luminescent performance of functionalized SBA-15 hybrids was more favorable than that of functionalized SBA-16 hybrids, revealing that SBA-15 was a better host material for lanthanide complex than mesoporous silica SBA-16.

  14. What are the barriers which discourage 15-16 year-old girls from participating in team sports and how can we overcome them?

    PubMed

    Wetton, Abigail R; Radley, Rebecca; Jones, Angela R; Pearce, Mark S

    2013-01-01

    Given the clear benefits of regular physical activity (such as reduced risks of cardiovascular disease and obesity, as well as other benefits including those related to mental health), exploration of the reasons that adolescent girls give for not taking part in team sports may be particularly valuable for enhancing later rates of participation. We combined questionnaires (n = 60) and semistructured interviews (n = 6) to assess the barriers that prevent 15-16-year-old girls from participating in extracurricular team games and what can be done to overcome these barriers and improve physical activity levels. Four barriers became prominent as to why girls in this sample do not participate: Internal Factors, Existing Stereotypes, Other Hobbies and Teachers. Methods to overcome these barriers were identified; changing teachers' attitudes and shifting the media's focus away from male sport. Following the successful summer Olympics and Paralympics in the UK, and the resulting positive focus on some of the nation's female athletes, a shift in focus may be possible. However, this needs to be maintained to allow girls more opportunities, role models and motivation to participate in sport.

  15. Lymphocyte subpopulations in the liver, spleen, intestines, and mesenteric nodes: an immunohistochemical study using human fetuses at 15-16 weeks.

    PubMed

    Hwang, Si Eun; Kim, Ji Hyun; Yu, Hee Chul; Murakami, Gen; Cho, Baik Hwan

    2014-08-01

    The roles of the liver and intestines in lymphocyte differentiation in human fetuses were assessed by immunohistochemical analysis of the thymus, bone marrow, liver, spleen, intestines, and lymph nodes of 15-16 week human fetuses using primary antibodies against IgM, CD3, CD7, CD8, CD10, CD20, CD45RO, HLA-DR, and CD68. The density of immunoreactive lymphocytes was high in the thymus and lymph nodes, but much lower in the bones, liver, spleen, and intestines. The medulla of the thymus contained IgM-positive mature B lymphocytes as well as CD20-positve B lymphocytes. In contrast, CD10-positive immature B lymphocytes were restricted in the cortex. There were no site-dependent differences among axillary, mediastinal, mesenteric, and pelvic lymph nodes. CD68-positive cells were observed at all sites examined. Many HLA-DR-positive round cells were present in the thymus, with fewer in the liver and spleen. The absolute number of lymphocytes was estimated to be ≥10-fold higher in lymph nodes than in liver. Although limited by analysis of only one fetal stage, these findings suggest that mesenteric nodes are likely to be more important than the liver, spleen, and intestines for lymphocyte proliferation and differentiation in human mid-term fetuses.

  16. What Are the Barriers Which Discourage 15-16 Year-Old Girls from Participating in Team Sports and How Can We Overcome Them?

    PubMed Central

    Wetton, Abigail R.; Jones, Angela R.; Pearce, Mark S.

    2013-01-01

    Given the clear benefits of regular physical activity (such as reduced risks of cardiovascular disease and obesity, as well as other benefits including those related to mental health), exploration of the reasons that adolescent girls give for not taking part in team sports may be particularly valuable for enhancing later rates of participation. We combined questionnaires (n = 60) and semistructured interviews (n = 6) to assess the barriers that prevent 15-16-year-old girls from participating in extracurricular team games and what can be done to overcome these barriers and improve physical activity levels. Four barriers became prominent as to why girls in this sample do not participate: Internal Factors, Existing Stereotypes, Other Hobbies and Teachers. Methods to overcome these barriers were identified; changing teachers' attitudes and shifting the media's focus away from male sport. Following the successful summer Olympics and Paralympics in the UK, and the resulting positive focus on some of the nation's female athletes, a shift in focus may be possible. However, this needs to be maintained to allow girls more opportunities, role models and motivation to participate in sport. PMID:24073416

  17. Disentangling Sources of Selection on Exonic Transcriptional Enhancers.

    PubMed

    Agoglia, Rachel M; Fraser, Hunter B

    2016-02-01

    In addition to coding for proteins, exons can also impact transcription by encoding regulatory elements such as enhancers. It has been debated whether such features confer heightened selective constraint, or evolve neutrally. We have addressed this question by developing a new approach to disentangle the sources of selection acting on exonic enhancers, in which we model the evolutionary rates of every possible substitution as a function of their effects on both protein sequence and enhancer activity. In three exonic enhancers, we found no significant association between evolutionary rates and effects on enhancer activity. This suggests that despite having biochemical activity, these exonic enhancers have no detectable selective constraint, and thus are unlikely to play a major role in protein evolution.

  18. Thermoelectric properties of homogeneously and non-homogeneously doped CdTe15/16M1/16 (M=N, P, As, Sb) and Cd15/16TeM1/16 (M=Na, K, Rb, Cs)

    NASA Astrophysics Data System (ADS)

    Yang, X. H.; Qin, X. Y.; Li, D.; Zhang, J.; Song, C. J.; Liu, Y. F.; Wang, L.; Xin, H. X.

    2015-11-01

    The electrical transport properties of p-doped semiconductors CdTe15/16M1/16 (M=N, P, As, Sb) and Cd15/16TeM1/16 (M=Na, K, Rb, Cs) with two configurations are investigated through first-principles calculations combined with Boltzmann transport theory under the relaxation time approximation. It is found that N and Cs atoms in the homogeneous structure induce much sharper electron densities of states (DOSs) and flatter energy bands at the valence band edges than the rest of doped elements, resulting in much larger Seebeck coefficients. The calculations reveal that most of the Seebeck coefficients and electrical conductivities are impacted unfavorably by the conglomeration of impurity atoms considered. Though the power factors for homogeneous doping of N and Cs are comparatively smaller, the electronic figures of merit are much larger at 800-1000 K than the rest ones due to much smaller electronic thermal conductivities, therefore probably enhancing the thermoelectric figures of merit. The results show that doping the elements with electronegativities distinct from the host atoms can enhance the Seebeck coefficients and the thermoelectric performances of bulk semiconductors efficiently if the energy levels of doped atoms resonate with those of host atoms and the arrangement of doped atoms is modulated appropriately to avoid deteriorating the sharpness of the DOS (or transport distribution).

  19. A case of Becker muscular dystrophy resulting from the skipping of four contiguous exons (71-74) of the dystrophin gene during mRNA maturation.

    PubMed

    Patria, S Y; Alimsardjono, H; Nishio, H; Takeshima, Y; Nakamura, H; Matsuo, M

    1996-07-01

    The mutations in one-third of both Duchenne and Becker muscular dystrophy patients remain unknown because they do not involve gross rearrangements of the dystrophin gene. Here we report the first example of multiple exon skipping during the splicing of dystrophin mRNA precursor encoded by an apparently normal dystrophin gene. A 9-year-old Japanese boy exhibiting excessive fatigue and high serum creatine kinase activity was examined for dystrophinopathy. An immunohistochemical study of muscle tissue biopsy disclosed faint and discontinuous staining of the N-terminal and rod domains of dystrophin but no staining at all of the C-terminal domain of dystrophin. The dystrophin transcript from muscle tissue was analyzed by the reverse transcriptase polymerase chain reaction. An amplified product encompassing exons 67-79 of dystrophin cDNA was found to be smaller than that of the wild-type product. Sequence analysis of this fragment showed that the 3' end of exon 70 was directly connected to the 5' end of exon 75 and, thus, that exons 71-74 were completely absent. As a result, a truncated dystrophin protein lacking 110 amino acids from the C-terminal domain should result from translation of this truncated mRNA, and the patient was diagnosed as having Becker muscular dystrophy at the molecular level. Genomic DNA was analyzed to identify the cause of the disappearance of these exons. Every exon-encompassing region could be amplified from genomic DNA, indicating that the dystrophin gene is intact. Furthermore, sequencing of these amplified products did not disclose any particular nucleotide change that could be responsible for the multiple exon skipping observed. Considering that exons 71-74 are spliced out alternatively in some tissue-specific isoforms, to suppose that the alternative splicing machinery is present in the muscle tissue of the index case and that it is activated by an undetermined mechanism is reasonable. These results illustrate a novel genetic anomaly that

  20. Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

    PubMed

    Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

    2015-01-01

    In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. PMID:26663798

  1. Short Exon Detection via Wavelet Transform Modulus Maxima

    PubMed Central

    Zhang, Xiaolei; Shen, Zhiwei; Zhang, Guishan; Shen, Yuanyu; Chen, Miaomiao; Zhao, Jiaxiang; Wu, Renhua

    2016-01-01

    The detection of short exons is a challenging open problem in the field of bioinformatics. Due to the fact that the weakness of existing model-independent methods lies in their inability to reliably detect small exons, a model-independent method based on the singularity detection with wavelet transform modulus maxima has been developed for detecting short coding sequences (exons) in eukaryotic DNA sequences. In the analysis of our method, the local maxima can capture and characterize singularities of short exons, which helps to yield significant patterns that are rarely observed with the traditional methods. In order to get some information about singularities on the differences between the exon signal and the background noise, the noise level is estimated by filtering the genomic sequence through a notch filter. Meanwhile, a fast method based on a piecewise cubic Hermite interpolating polynomial is applied to reconstruct the wavelet coefficients for improving the computational efficiency. In addition, the output measure of a paired-numerical representation calculated in both forward and reverse directions is used to incorporate a useful DNA structural property. The performances of our approach and other techniques are evaluated on two benchmark data sets. Experimental results demonstrate that the proposed method outperforms all assessed model-independent methods for detecting short exons in terms of evaluation metrics. PMID:27635656

  2. Short Exon Detection via Wavelet Transform Modulus Maxima.

    PubMed

    Zhang, Xiaolei; Shen, Zhiwei; Zhang, Guishan; Shen, Yuanyu; Chen, Miaomiao; Zhao, Jiaxiang; Wu, Renhua

    2016-01-01

    The detection of short exons is a challenging open problem in the field of bioinformatics. Due to the fact that the weakness of existing model-independent methods lies in their inability to reliably detect small exons, a model-independent method based on the singularity detection with wavelet transform modulus maxima has been developed for detecting short coding sequences (exons) in eukaryotic DNA sequences. In the analysis of our method, the local maxima can capture and characterize singularities of short exons, which helps to yield significant patterns that are rarely observed with the traditional methods. In order to get some information about singularities on the differences between the exon signal and the background noise, the noise level is estimated by filtering the genomic sequence through a notch filter. Meanwhile, a fast method based on a piecewise cubic Hermite interpolating polynomial is applied to reconstruct the wavelet coefficients for improving the computational efficiency. In addition, the output measure of a paired-numerical representation calculated in both forward and reverse directions is used to incorporate a useful DNA structural property. The performances of our approach and other techniques are evaluated on two benchmark data sets. Experimental results demonstrate that the proposed method outperforms all assessed model-independent methods for detecting short exons in terms of evaluation metrics. PMID:27635656

  3. Simultaneous scoring of 10 chromosomes (9,13,14,15,16,18,21,22,X, and Y) in interphase nuclei by using spectral imaging

    NASA Astrophysics Data System (ADS)

    Fung, Jingly; Weier, Heinz-Ulli G.; Goldberg, James D.; Pedersen, Roger A.

    1999-06-01

    Numerical aberrations involving parts of or entire chromosomes have detrimental effects on mammalian embryonic, and perinatal development. Only few fetuses with chromosomal imbalances survive to term, and their abnormalities lead to stillbirth or cause severely altered phenotypes in the offspring (such as trisomies involving chromosomes 13, 18, 21, and anomalies of X, and Y). Because aneuploidy of any of the 24 chromosomes will have significant consequences, an optimized preimplantation and prenatal genetic diagnosis (PGD) test will score all the chromosomes. Since most cells to be analyzed will be in interphase rather than metaphase, we developed a rapid procedure for the analysis of interphase cells such as lymphocytes, amniocytes, or early embryonic cells (blastomeres). Our approach was based on in situ hybridization of chromosome-specific non-isotopically labeled DNA probes and Spectral Imaging. The Spectral Imaging system uses an interferometer instead of standard emission filters in a fluorescence microscope to record high resolution spectra from fluorescently stained specimens. This bio-imaging system combines the techniques of fluorescence optical microscopy, charged coupled device imaging, Fourier spectroscopy, light microscopy, and powerful analysis software. The probe set used here allowed simultaneous detection of 10 chromosomes (9, 13, 14, 15, 16, 18, 21, 22, X, Y) in interphase nuclei. Probes were obtained commercially or prepared in-house. Following 16 - 40 h hybridization to interphase cells and removal of unbound probes, image spectra (range 450 - 850 nm, resolution 10 nm) were recorded and analyzed using an SD200 Spectral Imaging system (ASI, Carlsbad, CA). Initially some amniocytes were unscoreable due to their thickness, and fixation protocols had to be modified to achieve satisfactory results. In summary, this study shows the simultaneous detection of at least 10 different chromosomes in interphase cells using a novel approach for multi

  4. Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    PubMed Central

    Redpath, G. M. I.; Woolger, N.; Piper, A. K.; Lemckert, F. A.; Lek, A.; Greer, P. A.; North, K. N.; Cooper, S. T.

    2014-01-01

    Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a–specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module. PMID:25143396

  5. Functional and Structural Consequence of Rare Exonic Single Nucleotide Polymorphisms: One Story, Two Tales.

    PubMed

    Gu, Wanjun; Gurguis, Christopher I; Zhou, Jin J; Zhu, Yihua; Ko, Eun-A; Ko, Jae-Hong; Wang, Ting; Zhou, Tong

    2015-10-01

    Genetic variation arising from single nucleotide polymorphisms (SNPs) is ubiquitously found among human populations. While disease-causing variants are known in some cases, identifying functional or causative variants for most human diseases remains a challenging task. Rare SNPs, rather than common ones, are thought to be more important in the pathology of most human diseases. We propose that rare SNPs should be divided into two categories dependent on whether the minor alleles are derived or ancestral. Derived alleles are less likely to have been purified by evolutionary processes and may be more likely to induce deleterious effects. We therefore hypothesized that the rare SNPs with derived minor alleles would be more important for human diseases and predicted that these variants would have larger functional or structural consequences relative to the rare variants for which the minor alleles are ancestral. We systematically investigated the consequences of the exonic SNPs on protein function, mRNA structure, and translation. We found that the functional and structural consequences are more significant for the rare exonic variants for which the minor alleles are derived. However, this pattern is reversed when the minor alleles are ancestral. Thus, the rare exonic SNPs with derived minor alleles are more likely to be deleterious. Age estimation of rare SNPs confirms that these potentially deleterious SNPs are recently evolved in the human population. These results have important implications for understanding the function of genetic variations in human exonic regions and for prioritizing functional SNPs in genome-wide association studies of human diseases. PMID:26454016

  6. Functional and Structural Consequence of Rare Exonic Single Nucleotide Polymorphisms: One Story, Two Tales

    PubMed Central

    Gu, Wanjun; Gurguis, Christopher I.; Zhou, Jin J.; Zhu, Yihua; Ko, Eun-A.; Ko, Jae-Hong; Wang, Ting; Zhou, Tong

    2015-01-01

    Genetic variation arising from single nucleotide polymorphisms (SNPs) is ubiquitously found among human populations. While disease-causing variants are known in some cases, identifying functional or causative variants for most human diseases remains a challenging task. Rare SNPs, rather than common ones, are thought to be more important in the pathology of most human diseases. We propose that rare SNPs should be divided into two categories dependent on whether the minor alleles are derived or ancestral. Derived alleles are less likely to have been purified by evolutionary processes and may be more likely to induce deleterious effects. We therefore hypothesized that the rare SNPs with derived minor alleles would be more important for human diseases and predicted that these variants would have larger functional or structural consequences relative to the rare variants for which the minor alleles are ancestral. We systematically investigated the consequences of the exonic SNPs on protein function, mRNA structure, and translation. We found that the functional and structural consequences are more significant for the rare exonic variants for which the minor alleles are derived. However, this pattern is reversed when the minor alleles are ancestral. Thus, the rare exonic SNPs with derived minor alleles are more likely to be deleterious. Age estimation of rare SNPs confirms that these potentially deleterious SNPs are recently evolved in the human population. These results have important implications for understanding the function of genetic variations in human exonic regions and for prioritizing functional SNPs in genome-wide association studies of human diseases. PMID:26454016

  7. Macaca specific exon creation event generates a novel ZKSCAN5 transcript.

    PubMed

    Kim, Young-Hyun; Choe, Se-Hee; Song, Bong-Seok; Park, Sang-Je; Kim, Myung-Jin; Park, Young-Ho; Yoon, Seung-Bin; Lee, Youngjeon; Jin, Yeung Bae; Sim, Bo-Woong; Kim, Ji-Su; Jeong, Kang-Jin; Kim, Sun-Uk; Lee, Sang-Rae; Park, Young-Il; Huh, Jae-Won; Chang, Kyu-Tae

    2016-02-15

    ZKSCAN5 (also known as ZFP95) is a zinc-finger protein belonging to the Krűppel family. ZKSCAN5 contains a SCAN box and a KRAB A domain and is proposed to play a distinct role during spermatogenesis. In humans, alternatively spliced ZKSCAN5 transcripts with different 5'-untranslated regions (UTRs) have been identified. However, investigation of our Macaca UniGene Database revealed novel alternative ZKSCAN5 transcripts that arose due to an exon creation event. Therefore, in this study, we identified the full-length sequences of ZKSCAN5 and its alternative transcripts in Macaca spp. Additionally, we investigated different nonhuman primate sequences to elucidate the molecular mechanism underlying the exon creation event. We analyzed the evolutionary features of the ZKSCAN5 transcripts by reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR, and by sequencing various nonhuman primate DNA and RNA samples. The exon-created transcript was only detected in the Macaca lineage (crab-eating monkey and rhesus monkey). Full-length sequence analysis by rapid amplification of cDNA ends (RACE) identified ten full-length transcripts and four functional isoforms of ZKSCAN5. Protein sequence analyses revealed the presence of two groups of isoforms that arose because of differences in start-codon usage. Together, our results demonstrate that there has been specific selection for a discrete set of ZKSCAN5 variants in the Macaca lineage. Furthermore, study of this locus (and perhaps others) in Macaca spp. might facilitate our understanding of the evolutionary pressures that have shaped the mechanism of exon creation in primates. PMID:26657034

  8. Lamin A Δexon9 mutation leads to telomere and chromatin defects but not genomic instability

    PubMed Central

    Das, Arindam; Grotsky, David A; Neumann, Martin A; Kreienkamp, Ray; Gonzalez-Suarez, Ignacio; Redwood, Abena B; Kennedy, Brian K; Stewart, Colin L; Gonzalo, Susana

    2013-01-01

    Over 300 mutations in the LMNA gene, encoding A-type lamins, are associated with 15 human degenerative disorders and premature aging syndromes. Although genomic instability seems to contribute to the pathophysiology of some laminopathies, there is limited information about what mutations cause genomic instability and by which molecular mechanisms. Mouse embryonic fibroblasts depleted of A-type lamins or expressing mutants lacking exons 8–11 (LmnaΔ8–11/Δ8–11) exhibit alterations in telomere biology and DNA repair caused by cathepsin L-mediated degradation of 53BP1 and reduced expression of BRCA1 and RAD51. Thus, a region encompassing exons 8–11 seems essential for genome integrity. Given that deletion of lamin A exon 9 in the mouse (LmnaΔ9/Δ9) results in a progeria phenotype, we tested if this domain is important for genome integrity. LmnaΔ9/Δ9 MEFs exhibit telomere shortening and heterochromatin alterations but do not activate cathepsin L-mediated degradation of 53BP1 and maintain expression of BRCA1 and RAD51. Accordingly, LmnaΔ9/Δ9 MEFs do not present genomic instability, and expression of mutant lamin A Δexon9 in lamin-depleted cells restores DNA repair factors levels and partially rescues nuclear abnormalities. These data reveal that the domain encoded by exon 9 is important to maintain telomere homeostasis and heterochromatin structure but does not play a role in DNA repair, thus pointing to other exons in the lamin A tail as responsible for the genomic instability phenotype in LmnaΔ8–11/Δ8–11 mice. Our study also suggests that the levels of DNA repair factors 53BP1, BRCA1 and RAD51 could potentially serve as biomarkers to identify laminopathies that present with genomic instability. PMID:24153156

  9. NR4A3, a possibile oncogenic factor for neuroblastoma associated with CpGi methylation within the third exon

    PubMed Central

    UEKUSA, SHOTA; KAWASHIMA, HIROYUKI; SUGITO, KIMINOBU; YOSHIZAWA, SHINSUKE; SHINOJIMA, YUI; IGARASHI, JUN; GHOSH, SRIMOYEE; WANG, XAOFEI; FUJIWARA, KYOKO; IKEDA, TARO; KOSHINAGA, TSUGUMICHI; SOMA, MASAYOSHI; NAGASE, HIROKI

    2014-01-01

    Aberrant methylation of Nr4a3 exon 3 CpG island (CpGi) was initially identified during multistep mouse skin carcinogenesis. Nr4a3 is also known as a critical gene for neuronal development. Thus, we examined the Nr4a3 exon 3 CpGi methylation in mouse brain tissues from 15-day embryos, newborns and 12-week-old adults and found significant increase of its methylation and Nr4a3 expression during mouse brain development after birth. In addition, homologous region in human genome was frequently and aberrantly methylated in neuroblastoma specimens. A quantitative analysis of DNA methylation revealed that hypomethylation of CpG islands on NR4A3 exon 3, but not on exon 1 was identified in three neuroblastomas compared with matched adrenal glands. Additional analysis for 20 neuroblastoma patients was performed and 8 of 20 showed hypomethylation of the CpGi on NR4A3 exon 3. The survival rate of those 8 patients was significantly lower compared with those in patients with hypermethylation. Immunohistochemical NR4A3 expression was generally faint in neuroblastoma tissues compared with normal tissues. Moreover, the MYCN amplified NB9 cell line showed hypomethylation and low expression of NR4A3, while the non-MYCN amplified NB69 cell line showed hypermethylation and high expression. These results indicate that DNA hypomethylation of the CpGi at NR4A3 exon 3 is associated with low NR4A3 expression, and correlates with poor prognosis of neuroblastoma. Since NR4A3 upregulation associated with the hypermethylation and neuronal differentiation in mice, poor prognosis of neuroblastoma associated with NR4A3 low expression may be partly explained by dysregulation of its differentiation. PMID:24626568

  10. Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene

    SciTech Connect

    Christiano, A.M.; Chung-Honet, L.C.; Greenspan, D.S.; Hoffman, G.G.; Lee, S.; Cheng, W. ); Uitto, J. )

    1994-05-01

    The human type VII collagen (COL7A1) gene is the locus for mutations in at least some cases of dystrophic epidermolysis bullosa. Here the authors describe the entire intron/exon organization of COL7A1, which is shown to have 118 exons, more than any previously described gene. Despite this complexity, COL7A1 is compact. Consisting of 31,132 bp from transcription start site to polyadenylation site, it is only about three times the size of type VII collagen mRNA. Thus, COL7A1 introns are small. A 71-nucleotide COL7A1 intron is the smallest intron yet reported in a collagen gene, and only one COL7A1 intron is greater than 1 kb in length. All exons in the COL7A1 triple helix coding region that do not begin with sequences corresponding to imperfections of the triple helix begin with intact codons for Gly residues of Gly-X-Y repeats. This is reminiscent of the structure of fibrillar rather than other nonfibrillar collagen genes. In addition, the COL7A1 triple helix coding region contains many exons of recurring sizes (e.g., 25 exons are 36 bp, 12 exons are 45 bp, 8 exons are 63 bp), suggesting an evolutionary origin distinct from those of other nonfibrillar collagen genes. Sequences from the 5[prime] portion of COL7A1 are presented along with the 3766-bp intergenic sequence, which separated COL7A1 from the upstream gene encoding the core I protein of the cytochrome bc[sub 1] complex. The COL7A1 promoter region is found to lack extensive homologies with promoter regions of other genes expressed primarily in skin. 60 refs., 5 figs., 1 tab.

  11. Intron Retention and TE Exonization Events in ZRANB2

    PubMed Central

    Park, Sang-Je; Huh, Jae-Won; Kim, Young-Hyun; Kim, Heui-Soo; Chang, Kyu-Tae

    2012-01-01

    The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2), contains arginine/serine-rich (RS) domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat_L3) between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat_L3). Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species). Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat_L3). RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys) and were expressed via intron retention (IR). Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs) exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution. PMID:22778693

  12. The evolution of the coding exome of the Arabidopsis species - the influences of DNA methylation, relative exon position, and exon length

    PubMed Central

    2014-01-01

    Background The evolution of the coding exome is a major driving force of functional divergence both between species and between protein isoforms. Exons at different positions in the transcript or in different transcript isoforms may (1) mutate at different rates due to variations in DNA methylation level; and (2) serve distinct biological roles, and thus be differentially targeted by natural selection. Furthermore, intrinsic exonic features, such as exon length, may also affect the evolution of individual exons. Importantly, the evolutionary effects of these intrinsic/extrinsic features may differ significantly between animals and plants. Such inter-lineage differences, however, have not been systematically examined. Results Here we examine how DNA methylation at CpG dinucleotides (CpG methylation), in the context of intrinsic exonic features (exon length and relative exon position in the transcript), influences the evolution of coding exons of Arabidopsis thaliana. We observed fairly different evolutionary patterns in A. thaliana as compared with those reported for animals. Firstly, the mutagenic effect of CpG methylation is the strongest for internal exons and the weakest for first exons despite the stringent selective constraints on the former group. Secondly, the mutagenic effect of CpG methylation increases significantly with length in first exons but not in the other two exon groups. Thirdly, CpG methylation level is correlated with evolutionary rates (dS, dN, and the dN/dS ratio) with markedly different patterns among the three exon groups. The correlations are generally positive, negative, and mixed for first, last, and internal exons, respectively. Fourthly, exon length is a CpG methylation-independent indicator of evolutionary rates, particularly for dN and the dN/dS ratio in last and internal exons. Finally, the evolutionary patterns of coding exons with regard to CpG methylation differ significantly between Arabidopsis species and mammals. Conclusions

  13. Deletion of exon 8 from the EXT1 gene causes multiple osteochondromas (MO) in a family with three affected members.

    PubMed

    Zhuang, Lei; Gerber, Simon D; Kuchen, Stefan; Villiger, Peter M; Trueb, Beat

    2016-01-01

    Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

  14. PCA3 gene expression in prostate cancer tissue in a Chinese population: quantification by real-time FQ-RT-PCR based on exon 3 of PCA3.

    PubMed

    Tao, Zhihua; Shen, Mo; Zheng, Yanbo; Mao, Xiaolu; Chen, Zhanguo; Yin, Yibing; Yu, Kaiyuan; Weng, Zhiliang; Xie, Hui; Li, Chengdi; Wu, Xiuling; Hu, Yuanping; Zhang, Xiaohua; Wang, Ouchen; Song, Qitong; Yu, Zhixian

    2010-08-01

    Prostate cancer (PCa) is the second most common cancer in men, and its incidence is still increasing. PCA3 is the most prostate cancer specific biomarker. Here we confirmed that both exon 3 and exon 4 are in the prostate-specific region of the PCA3 gene, and established the methodology of real-time fluorescent quantitative RT-PCR (FQ-RT-PCR) detecting PCA3 mRNA with primer spanning exons 1 and 3, and evaluated its clinical utility in a Chinese population. What disclosed that PCA3 mRNA is prostate cancer specific and shows increased expression in prostate cancer. It could be a reliable molecular marker in prostate cancer diagnosis. Exon 3-based real-time FQ-RT-PCR may prove useful in prostate cancer diagnosis, given that the associated primer would span only exons 1 and 3, relative to other models spanning exons 1 to 4. A shorter amplicon would not only enhance the efficiency of real-time FQ-RT-PCR, but may also simplify the quantification of PCA3 mRNA.

  15. Evolution of the Exon-Intron Structure in Ciliate Genomes.

    PubMed

    Bondarenko, Vladyslav S; Gelfand, Mikhail S

    2016-01-01

    A typical eukaryotic gene is comprised of alternating stretches of regions, exons and introns, retained in and spliced out a mature mRNA, respectively. Although the length of introns may vary substantially among organisms, a large fraction of genes contains short introns in many species. Notably, some Ciliates (Paramecium and Nyctotherus) possess only ultra-short introns, around 25 bp long. In Paramecium, ultra-short introns with length divisible by three (3n) are under strong evolutionary pressure and have a high frequency of in-frame stop codons, which, in the case of intron retention, cause premature termination of mRNA translation and consequent degradation of the mis-spliced mRNA by the nonsense-mediated decay mechanism. Here, we analyzed introns in five genera of Ciliates, Paramecium, Tetrahymena, Ichthyophthirius, Oxytricha, and Stylonychia. Introns can be classified into two length classes in Tetrahymena and Ichthyophthirius (with means 48 bp, 69 bp, and 55 bp, 64 bp, respectively), but, surprisingly, comprise three distinct length classes in Oxytricha and Stylonychia (with means 33-35 bp, 47-51 bp, and 78-80 bp). In most ranges of the intron lengths, 3n introns are underrepresented and have a high frequency of in-frame stop codons in all studied species. Introns of Paramecium, Tetrahymena, and Ichthyophthirius are preferentially located at the 5' and 3' ends of genes, whereas introns of Oxytricha and Stylonychia are strongly skewed towards the 5' end. Analysis of evolutionary conservation shows that, in each studied genome, a significant fraction of intron positions is conserved between the orthologs, but intron lengths are not correlated between the species. In summary, our study provides a detailed characterization of introns in several genera of Ciliates and highlights some of their distinctive properties, which, together, indicate that splicing spellchecking is a universal and evolutionarily conserved process in the biogenesis of short introns in

  16. Evolution of the Exon-Intron Structure in Ciliate Genomes

    PubMed Central

    Gelfand, Mikhail S.

    2016-01-01

    A typical eukaryotic gene is comprised of alternating stretches of regions, exons and introns, retained in and spliced out a mature mRNA, respectively. Although the length of introns may vary substantially among organisms, a large fraction of genes contains short introns in many species. Notably, some Ciliates (Paramecium and Nyctotherus) possess only ultra-short introns, around 25 bp long. In Paramecium, ultra-short introns with length divisible by three (3n) are under strong evolutionary pressure and have a high frequency of in-frame stop codons, which, in the case of intron retention, cause premature termination of mRNA translation and consequent degradation of the mis-spliced mRNA by the nonsense-mediated decay mechanism. Here, we analyzed introns in five genera of Ciliates, Paramecium, Tetrahymena, Ichthyophthirius, Oxytricha, and Stylonychia. Introns can be classified into two length classes in Tetrahymena and Ichthyophthirius (with means 48 bp, 69 bp, and 55 bp, 64 bp, respectively), but, surprisingly, comprise three distinct length classes in Oxytricha and Stylonychia (with means 33–35 bp, 47–51 bp, and 78–80 bp). In most ranges of the intron lengths, 3n introns are underrepresented and have a high frequency of in-frame stop codons in all studied species. Introns of Paramecium, Tetrahymena, and Ichthyophthirius are preferentially located at the 5' and 3' ends of genes, whereas introns of Oxytricha and Stylonychia are strongly skewed towards the 5' end. Analysis of evolutionary conservation shows that, in each studied genome, a significant fraction of intron positions is conserved between the orthologs, but intron lengths are not correlated between the species. In summary, our study provides a detailed characterization of introns in several genera of Ciliates and highlights some of their distinctive properties, which, together, indicate that splicing spellchecking is a universal and evolutionarily conserved process in the biogenesis of short introns in

  17. The role of short RNA loops in recognition of a single-hairpin exon derived from a mammalian-wide interspersed repeat

    PubMed Central

    Kralovicova, Jana; Patel, Alpa; Searle, Mark; Vorechovsky, Igor

    2015-01-01

    Splice-site selection is controlled by secondary structure through sequestration or approximation of splicing signals in primary transcripts but the exact role of even the simplest and most prevalent structural motifs in exon recognition remains poorly understood. Here we took advantage of a single-hairpin exon that was activated in a mammalian-wide interspersed repeat (MIR) by a mutation stabilizing a terminal triloop, with splice sites positioned close to each other in a lower stem of the hairpin. We first show that the MIR exon inclusion in mRNA correlated inversely with hairpin stabilities. Employing a systematic manipulation of unpaired regions without altering splice-site configuration, we demonstrate a high correlation between exon inclusion of terminal tri- and tetraloop mutants and matching tri-/tetramers in splicing silencers/enhancers. Loop-specific exon inclusion levels and enhancer/silencer associations were preserved across primate cell lines, in 4 hybrid transcripts and also in the context of a distinct stem, but only if its loop-closing base pairs were shared with the MIR hairpin. Unlike terminal loops, splicing activities of internal loop mutants were predicted by their intramolecular Watson-Crick interactions with the antiparallel strand of the MIR hairpin rather than by frequencies of corresponding trinucleotides in splicing silencers/enhancers. We also show that splicing outcome of oligonucleotides targeting the MIR exon depend on the identity of the triloop adjacent to their antisense target. Finally, we identify proteins regulating MIR exon recognition and reveal a distinct requirement of adjacent exons for C-terminal extensions of Tra2α and Tra2β RNA recognition motifs. PMID:25826413

  18. During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

    PubMed Central

    Bailleul, B

    1996-01-01

    Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping. PMID:8604331

  19. Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in mice

    NASA Technical Reports Server (NTRS)

    Donoho, Greg; Brenneman, Mark A.; Cui, Tracy X.; Donoviel, Dorit; Vogel, Hannes; Goodwin, Edwin H.; Chen, David J.; Hasty, Paul

    2003-01-01

    The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51. Copyright 2003 Wiley-Liss, Inc.

  20. The Levels of Tau Isoforms Containing Exon-2 and Exon-10 Segments Increased in the Cerebrospinal Fluids of the Patients with Sporadic Creutzfeldt-Jakob Disease.

    PubMed

    Chen, Cao; Zhou, Wei; Lv, Yan; Shi, Qi; Wang, Jing; Xiao, Kang; Chen, Li-Na; Zhang, Bao-Yun; Dong, Xiao-Ping

    2016-08-01

    The alteration of protein tau in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) has been widely evaluated, possessing a significant diagnostic value for CJD. With the biotin-labeled tau-exon-specific mAbs, direct ELISA methods were established and the levels of tau isoforms containing exon-2 and exon-10 segments in CSF of the patients with various human prion diseases and in brain tissues of scrapie-infected animals were evaluated. The results showed that the levels of tau, especially containing four repeats in microtubule binding domain, were increased in the CSF samples of the patients with sporadic CJD (sCJD). Using the unlabeled (cold) mixed exon-specific mAbs, a competitive tau ELISA was conducted based on a commercial tau kit. It revealed that the majority of the increased tau in the CSF of sCJD cases was derived from the tau isoforms with exon-2 and exon-10 segments. Increases of CSF tau isoforms with exon-2 and exon-10 segments were also observed in the patients of E200K and T188K genetic CJD (gCJD), but not in the cases of fatal familiar insomnia (FFI). The increasing levels of tau isoforms with exon-2 and exon-10 segments in the group of sCJD correlated well with the positive 14-3-3 in CSF. Additionally, the similar alterative profiles of tau isoforms with exon-2 and exon-10 segments were also observed in the brain tissues of scrapie-infected rodents and a sCJD patient. Our data here propose the tau isoforms with exon-2 and exon-10 segments increase in CSF of sCJD and some types of gCJD, which may help to understand the physiological metabolism and pathological significance of various tau isoforms in the pathogenesis of prion diseases.

  1. Widespread establishment and regulatory impact of Alu exons in human genes.

    PubMed

    Shen, Shihao; Lin, Lan; Cai, James J; Jiang, Peng; Kenkel, Elizabeth J; Stroik, Mallory R; Sato, Seiko; Davidson, Beverly L; Xing, Yi

    2011-02-15

    The Alu element has been a major source of new exons during primate evolution. Thousands of human genes contain spliced exons derived from Alu elements. However, identifying Alu exons that have acquired genuine biological functions remains a major challenge. We investigated the creation and establishment of Alu exons in human genes, using transcriptome profiles of human tissues generated by high-throughput RNA sequencing (RNA-Seq) combined with extensive RT-PCR analysis. More than 25% of Alu exons analyzed by RNA-Seq have estimated transcript inclusion levels of at least 50% in the human cerebellum, indicating widespread establishment of Alu exons in human genes. Genes encoding zinc finger transcription factors have significantly higher levels of Alu exonization. Importantly, Alu exons with high splicing activities are strongly enriched in the 5'-UTR, and two-thirds (10/15) of 5'-UTR Alu exons tested by luciferase reporter assays significantly alter mRNA translational efficiency. Mutational analysis reveals the specific molecular mechanisms by which newly created 5'-UTR Alu exons modulate translational efficiency, such as the creation or elongation of upstream ORFs that repress the translation of the primary ORFs. This study presents genomic evidence that a major functional consequence of Alu exonization is the lineage-specific evolution of translational regulation. Moreover, the preferential creation and establishment of Alu exons in zinc finger genes suggest that Alu exonization may have globally affected the evolution of primate and human transcriptomes by regulating the protein production of master transcriptional regulators in specific lineages.

  2. NextSearch: A Search Engine for Mass Spectrometry Data against a Compact Nucleotide Exon Graph.

    PubMed

    Kim, Hyunwoo; Park, Heejin; Paek, Eunok

    2015-07-01

    Proteogenomics research has been using six-frame translation of the whole genome or amino acid exon graphs to overcome the limitations of reference protein sequence database; however, six-frame translation is not suitable for annotating genes that span over multiple exons, and amino acid exon graphs are not convenient to represent novel splice variants and exon skipping events between exons of incompatible reading frames. We propose a proteogenomic pipeline NextSearch (Nucleotide EXon-graph Transcriptome Search) that is based on a nucleotide exon graph. This pipeline consists of constructing a compact nucleotide exon graph that systematically incorporates novel splice variations and a search tool that identifies peptides by directly searching the nucleotide exon graph against tandem mass spectra. Because our exon graph stores nucleotide sequences, it can easily represent novel splice variations and exon skipping events between incompatible reading frame exons. Searching for peptide identification is performed against this nucleotide exon graph, without converting it into a protein sequence in FASTA format, achieving an order of magnitude reduction in the size of the sequence database storage. NextSearch outputs the proteome-genome/transcriptome mapping results in a general feature format (GFF) file, which can be visualized by public tools such as the UCSC Genome Browser.

  3. Modular structural units, exons, and function in chicken lysozyme.

    PubMed Central

    Go, M

    1983-01-01

    By the application of the same algorithm for finding compact structural units encoded by exons as applied previously to hemoglobin, five units, M1-M5, were identified in chicken egg white lysozyme. They consist of residues 1-30, 31-55, 56-84, 85-108, and 109-129, respectively. I call these compact structural units "modules." As in hemoglobin, modules thus identified correspond well to exons--i.e., modules M1, M2 plus M3, M4, and M5 correspond to exons 1, 2, 3, and 4 of the lysozyme gene, respectively. Localization of the catalytic sites glutamic acid-35 and aspartic acid-52 on the module M2 suggests that this module might have worked as a functional unit in a primitive lysozyme. The good correspondence between exons and modules reinforces the idea of "proteins in pieces," which was derived from the fact of "genes in pieces." The evolutionary origin of the introns in globins and lysozyme is discussed. PMID:6572956

  4. Lineage-specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana.

    PubMed

    Bush, Stephen J; Kover, Paula X; Urrutia, Araxi O

    2015-06-01

    Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the 'edges' of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage-specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage-specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage-specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters.

  5. Lineage-specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana.

    PubMed

    Bush, Stephen J; Kover, Paula X; Urrutia, Araxi O

    2015-06-01

    Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the 'edges' of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage-specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage-specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage-specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters. PMID:25930165

  6. Alterations in exon 1 of c-myc and expression of p62c-myc in cervical squamous cell carcinoma.

    PubMed Central

    O'Leary, J J; Landers, R J; Crowley, M; Healy, I; Kealy, W F; Hogan, J; Doyle, C T

    1997-01-01

    AIMS: To examine human papillomavirus (HPV) positive and negative squamous cell carcinomas of the cervix for structural alterations in exon 1 c-myc; and to investigate the expression pattern of p62, the protein product of c-myc. MATERIAL: Archival paraffin wax embedded tissues of cervical squamous cell carcinomas, stage I and II, retrieved from the files of the department of pathology, University College Cork, Ireland: 40 cases were examined for alterations in exon 1 of c-myc; 57 cases were used for immunocytochemical p62 analysis. METHODS: c-myc exon 1 PCR on HPV positive and negative stage I and II cervical squamous cell carcinomas was performed using primers designed to fragile sites in exon 1 of the c-myc oncogene, which are frequently involved in translocation phenomena and deletions in other neoplasms. This region is bordered by two promoter sequences P1 and P2. In addition, the expression of p62 was evaluated using the monoclonal antibody Mycl-9E10. RESULTS: Alterations in exon 1 of c-myc were shown in 7.5% of squamous cell carcinomas of the cervix. Changes in exon 1 and 2 of c-myc were also found in COLO 320 cells and Raji cells. These alterations were due to small deletions within exon 1 of c-myc, but point polymorphisms occurring within the priming sites (in one case) may also have occurred. The alterations uncovered appeared "clonal," as replicate samples showed the same amplicon band pattern. Expression of c-myc was variable, with cytoplasmic staining patterns predominating. All cases which showed exon 1 alterations were HPV positive and had strong nuclear positivity on p62 immunocytochemistry. CONCLUSIONS: Alterations in exon 1 of c-myc occur in a minority of cervical cancers and there was increased expression of p62 in a cohort of HPV positive and negative cervical squamous cell carcinomas. Exon 1 alterations may provide an alternative route to c-myc activation in early squamous cell carcinoma. Images PMID:9462237

  7. A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping

    PubMed Central

    Walmsley, Gemma L.; Arechavala-Gomeza, Virginia; Fernandez-Fuente, Marta; Burke, Margaret M.; Nagel, Nicole; Holder, Angela; Stanley, Rachael; Chandler, Kate; Marks, Stanley L.; Muntoni, Francesco; Shelton, G. Diane; Piercy, Richard J.

    2010-01-01

    Background Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion “hot spot” is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD “hot spot”. Methodology/Principal Findings Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5′ donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. Conclusions/Significance Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD. PMID:20072625

  8. Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter

    SciTech Connect

    Yanagawa, H.; Nishio, H.; Takeshima, Y.

    1994-09-01

    The dystrophin gene, which is muted in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously-unidentified alternative promoter. The case study presented is that of patient with Duchenne muscular dystrophy who had a deletion extending from 5{prime} end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5{prime} part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5{prime} end of the new transcript indicated that the 5{prime} end of exon 3 was extended by 9 codons, only the last (most 3{prime}) of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5{prime} of the previously-identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

  9. THE RELATIONSHIP BETWEEN MOTOR COMPETENCE AND PHYSICAL FITNESS IS WEAKER IN THE 15-16 YR. ADOLESCENT AGE GROUP THAN IN YOUNGER AGE GROUPS (4-5 YR. AND 11-12 YR.).

    PubMed

    Haga, Monika; Gísladóttír, Thórdís; Sigmundsson, Hermundur

    2015-12-01

    Developing motor competence and physical fitness can affect the maintenance of a sufficient level of physical activity in children and adolescents. This study assesses the relationship between motor competence and physical fitness from childhood through early adolescence. A cross-sectional sample of 194 participants from 4 to 16 years old were divided into three groups; 4-6 yr. (n=42, M age=5.2, SD 0.6), 11-12 yr. (n=58, M age=12.4, SD=0.3), and 15-16 yr. (n=94, M age=15.9, SD=0.4). To assess motor competence, each child completed the Movement Assessment Battery for Children (MABC). To measure physical fitness, three tasks (strength, speed, and endurance) were selected from the Test of Physical Fitness (TPF). To analyze the significance of the difference between the correlation coefficient in the three age groups (samples) (4-6, 11-12, and 15-16 yr.), Fischer r-to-z transformation was used. The correlation (Pearson's) between motor competence and physical fitness in the age groups was statistically higher for the youngest age groups (4-6 and 11-12 yr.) and the adolescent group (age 15-16). The differences between the two youngest age groups were not statistically significant. The results demonstrate that the correlation between motor competence and physical fitness decreases with age. PMID:26595203

  10. Multiplex amplification of large sets of human exons.

    PubMed

    Porreca, Gregory J; Zhang, Kun; Li, Jin Billy; Xie, Bin; Austin, Derek; Vassallo, Sara L; LeProust, Emily M; Peck, Bill J; Emig, Christopher J; Dahl, Fredrik; Gao, Yuan; Church, George M; Shendure, Jay

    2007-11-01

    A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

  11. The IL-9 receptor gene (IL9R): Genomic structure, chromosomal localization in the pseudoautosomal region of the long arm of sex chromosomes, and identification of IL9R pseudogenes at 9qter, 10pter, 16pter, 18pter

    SciTech Connect

    Kermouni, A.; Godelaine, D.; Lurquin, C.; Szikora, J.P.

    1995-09-20

    Cosmids containing the human IL-9 receptor (R) gene (IL9R) have been isolated from a genomic library using the IL9R cDNA as a probe. We have shown that the human IL9R gene is composed of 11 exons and 10 introns, stretching over {approx} 17 kb, and is located within the pseudoautosomal region of the Xq and Yq chromosome, in the vicinity of the telomere. Analysis of the 5` flanking region revealed multiple transcription initiation sites as well as potential binding motifs for AP1, AP2, AP3, Sp1, and NF-kB, although this region lacks a TATA box. Using the human IL9R cosmid as a probe to perform fluorescence in situ hybridization, additional signals were identified in the subtelomeric regions of chromosomes 9q, 10p, 16p, and 18p. IL9R homologs located on chromosomes 9 and 18 were partially characterized, while those located on chromosomes 16 and 10 were completely sequenced. Although they are similiar to the IL9R gene ({approx} 90% identity), none of these copies encodes a functional receptor: none of them contains sequences homologous to the 5` flanking region or exon 1 of the IL9R gene, and the remaining ORFs have been inactivated by various point mutations and deletions. Taken together, our results indicate that the IL9R gene is located at Xq28 and Yq12, in the long arm pseudoautosomal region, and that four IL9R pseudogenes are located on 9q34, 10p15, 16p13.3 and 18p11.3, probably dispersed as the result of translocations during evolution. 42 refs., 6 figs., 3 tabs.

  12. HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform

    PubMed Central

    Nasrin, Farhana; Rahman, Mohammad Alinoor; Masuda, Akio; Ohe, Kenji; Takeda, Jun-ichi; Ohno, Kinji

    2014-01-01

    Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1. PMID:25354590

  13. Structural basis for exon recognition by a group II intron

    SciTech Connect

    Toor, Navtej; Rajashankar, Kanagalaghatta; Keating, Kevin S.; Pyle, Anna Marie

    2008-11-18

    Free group II introns are infectious retroelements that can bind and insert themselves into RNA and DNA molecules via reverse splicing. Here we report the 3.4-A crystal structure of a complex between an oligonucleotide target substrate and a group IIC intron, as well as the refined free intron structure. The structure of the complex reveals the conformation of motifs involved in exon recognition by group II introns.

  14. Isolation and characterisation of antibodies which specifically recognise the peptide encoded by exon 7 (v2) of the human CD44 gene

    PubMed Central

    Borgya, A; Woodman, A; Sugiyama, M; Donié, F; Kopetzki, E; Matsumura, Y; Tarin, D

    1995-01-01

    Aims—Exon 7 of the human CD44 gene is overexpressed in many commonly occurring carcinomas. The aim of the study was to explore the diagnostic and therapeutic potential of this frequent abnormality. Methods—A new monoclonal antibody (mAb, M-23.6.1) and a polyclonal antibody (pAb,S-6127) to the corresponding antigen were raised by immunising mice and sheep, respectively, with a specially constructed fusion protein HIV2 (gp32)-CD44 exon 7. Results—Characterisation of mAb, M-23.6.1 by ELISA, western blotting, immunocytochemistry, and FACS analysis confirmed that it specifically recognises an epitope in the region between amino acids 19 and 33 of the peptide encoded by this exon. Western blotting experiments with two cell lines, RT112 and ZR75-1, known from RT-PCR data to be overtranscribing the exon, yielded a monospecific band of approximately 220 kDa, and immunocytochemistry showed discrete membrane staining on the same cell lines. Fluorescent antibody cell sorting (FACS) revealed binding to greater than 90% of the cells of each of these lines. Specificity of recognition of the antigen was shown by inhibition of the precise immunoreactivity typically seen in ELISA and Western blots, by pre-incubation with synthetic exon 7 peptide or fragments of it. Conclusions—The new antibodies will be useful tools for the further analysis of abnormal CD44 isoforms and their clinical implications. Images PMID:16696015

  15. Gain of a New Exon by a Lineage-Specific Alu Element-Integration Event in the BCS1L Gene during Primate Evolution

    PubMed Central

    Park, Sang-Je; Kim, Young-Hyun; Lee, Sang-Rae; Choe, Se-Hee; Kim, Myung-Jin; Kim, Sun-Uk; Kim, Ji-Su; Sim, Bo-Woong; Song, Bong-Seok; Jeong, Kang-Jin; Jin, Yeung-Bae; Lee, Youngjeon; Park, Young-Ho; Park, Young Il; Huh, Jae-Won; Chang, Kyu-Tae

    2015-01-01

    BCS1L gene encodes mitochondrial protein and is a member of conserved AAA protein family. This gene is involved in the incorporation of Rieske FeS and Qcr10p into complex III of respiratory chain. In our previous study, AluYRa2-derived alternative transcript in rhesus monkey genome was identified. However, this transcript has not been reported in human genome. In present study, we conducted evolutionary analysis of AluYRa2-exonized transcript with various primate genomic DNAs and cDNAs from humans, rhesus monkeys, and crab-eating monkeys. Remarkably, our results show that AluYRa2 element has only been integrated into genomes of Macaca species. This Macaca lineage-specific integration of AluYRa2 element led to exonization event in the first intron region of BCS1L gene by producing a conserved 3′ splice site. Intriguingly, in rhesus and crab-eating monkeys, more diverse transcript variants by alternative splicing (AS) events, including exon skipping and different 5′ splice sites from humans, were identified. Alignment of amino acid sequences revealed that AluYRa2-exonized transcript has short N-terminal peptides. Therefore, AS events play a major role in the generation of various transcripts and proteins during primate evolution. In particular, lineage-specific integration of Alu elements and species-specific Alu-derived exonization events could be important sources of gene diversification in primates. PMID:26537194

  16. Targeted Exon Sequencing in Usher Syndrome Type I

    PubMed Central

    Bujakowska, Kinga M.; Consugar, Mark; Place, Emily; Harper, Shyana; Lena, Jaclyn; Taub, Daniel G.; White, Joseph; Navarro-Gomez, Daniel; Weigel DiFranco, Carol; Farkas, Michael H.; Gai, Xiaowu; Berson, Eliot L.; Pierce, Eric A.

    2014-01-01

    Purpose. Patients with Usher syndrome type I (USH1) have retinitis pigmentosa, profound congenital hearing loss, and vestibular ataxia. This syndrome is currently thought to be associated with at least six genes, which are encoded by over 180 exons. Here, we present the use of state-of-the-art techniques in the molecular diagnosis of a cohort of 47 USH1 probands. Methods. The cohort was studied with selective exon capture and next-generation sequencing of currently known inherited retinal degeneration genes, comparative genomic hybridization, and Sanger sequencing of new USH1 exons identified by human retinal transcriptome analysis. Results. With this approach, we were able to genetically solve 14 of the 47 probands by confirming the biallelic inheritance of mutations. We detected two likely pathogenic variants in an additional 19 patients, for whom family members were not available for cosegregation analysis to confirm biallelic inheritance. Ten patients, in addition to primary disease–causing mutations, carried rare likely pathogenic USH1 alleles or variants in other genes associated with deaf-blindness, which may influence disease phenotype. Twenty-one of the identified mutations were novel among the 33 definite or likely solved patients. Here, we also present a clinical description of the studied cohort at their initial visits. Conclusions. We found a remarkable genetic heterogeneity in the studied USH1 cohort with multiplicity of mutations, of which many were novel. No obvious influence of genotype on phenotype was found, possibly due to small sample sizes of the genotypes under study. PMID:25468891

  17. Variants Affecting Exon Skipping Contribute to Complex Traits

    PubMed Central

    Lee, Younghee; Gamazon, Eric R.; Rebman, Ellen; Lee, Yeunsook; Lee, Sanghyuk; Dolan, M. Eileen; Cox, Nancy J.; Lussier, Yves A.

    2012-01-01

    DNA variants that affect alternative splicing and the relative quantities of different gene transcripts have been shown to be risk alleles for some Mendelian diseases. However, for complex traits characterized by a low odds ratio for any single contributing variant, very few studies have investigated the contribution of splicing variants. The overarching goal of this study is to discover and characterize the role that variants affecting alternative splicing may play in the genetic etiology of complex traits, which include a significant number of the common human diseases. Specifically, we hypothesize that single nucleotide polymorphisms (SNPs) in splicing regulatory elements can be characterized in silico to identify variants affecting splicing, and that these variants may contribute to the etiology of complex diseases as well as the inter-individual variability in the ratios of alternative transcripts. We leverage high-throughput expression profiling to 1) experimentally validate our in silico predictions of skipped exons and 2) characterize the molecular role of intronic genetic variations in alternative splicing events in the context of complex human traits and diseases. We propose that intronic SNPs play a role as genetic regulators within splicing regulatory elements and show that their associated exon skipping events can affect protein domains and structure. We find that SNPs we would predict to affect exon skipping are enriched among the set of SNPs reported to be associated with complex human traits. PMID:23133393

  18. An Exon-Capture System for the Entire Class Ophiuroidea.

    PubMed

    Hugall, Andrew F; O'Hara, Timothy D; Hunjan, Sumitha; Nilsen, Roger; Moussalli, Adnan

    2016-01-01

    Exon-capture studies have typically been restricted to relatively shallow phylogenetic scales due primarily to hybridization constraints. Here, we present an exon-capture system for an entire class of marine invertebrates, the Ophiuroidea, built upon a phylogenetically diverse transcriptome foundation. The system captures approximately 90% of the 1,552 exon target, across all major lineages of the quarter-billion-year-old extant crown group. Key features of our system are 1) basing the target on an alignment of orthologous genes determined from 52 transcriptomes spanning the phylogenetic diversity and trimmed to remove anything difficult to capture, map, or align; 2) use of multiple artificial representatives based on ancestral state reconstructions rather than exemplars to improve capture and mapping of the target; 3) mapping reads to a multi-reference alignment; and 4) using patterns of site polymorphism to distinguish among paralogy, polyploidy, allelic differences, and sample contamination. The resulting data give a well-resolved tree (currently standing at 417 samples, 275,352 sites, 91% data-complete) that will transform our understanding of ophiuroid evolution and biogeography.

  19. Purifying Selection on Exonic Splice Enhancers in Intronless Genes

    PubMed Central

    Savisaar, Rosina; Hurst, Laurence D.

    2016-01-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  20. An Exon-Capture System for the Entire Class Ophiuroidea

    PubMed Central

    Hugall, Andrew F.; O’Hara, Timothy D.; Hunjan, Sumitha; Nilsen, Roger; Moussalli, Adnan

    2016-01-01

    Exon-capture studies have typically been restricted to relatively shallow phylogenetic scales due primarily to hybridization constraints. Here, we present an exon-capture system for an entire class of marine invertebrates, the Ophiuroidea, built upon a phylogenetically diverse transcriptome foundation. The system captures approximately 90% of the 1,552 exon target, across all major lineages of the quarter-billion-year-old extant crown group. Key features of our system are 1) basing the target on an alignment of orthologous genes determined from 52 transcriptomes spanning the phylogenetic diversity and trimmed to remove anything difficult to capture, map, or align; 2) use of multiple artificial representatives based on ancestral state reconstructions rather than exemplars to improve capture and mapping of the target; 3) mapping reads to a multi-reference alignment; and 4) using patterns of site polymorphism to distinguish among paralogy, polyploidy, allelic differences, and sample contamination. The resulting data give a well-resolved tree (currently standing at 417 samples, 275,352 sites, 91% data-complete) that will transform our understanding of ophiuroid evolution and biogeography. PMID:26474846

  1. Variation in the androgen receptor gene exon 1 CAG repeat correlates with manifestations of autoimmunity in women with lupus.

    PubMed

    Olsen, Nancy J; Benko, Ann L; Kovacs, William J

    2014-01-01

    Clinical and experimental evidence support a role for gonadal steroids in modulating the expression and course of autoimmune diseases such as lupus. Whether or not inherited variation in sensitivity to circulating androgenic hormones could influence the manifestations of such disease is, however, unknown. We sought to determine whether differences in androgen sensitivity conferred by variation in the exon 1 CAG repeat region of the androgen receptor (AR) gene were associated with differences in the clinical or humoral immune manifestations of lupus in a cohort of female subjects. We found that shorter AR CAG repeat lengths in lupus subjects correlated with a higher Systemic Lupus Erythematosus Disease Activity Index score, higher ANA levels, and expression of a broader array of IgG autoantibodies. Our findings of more severe clinical manifestations and more exuberant humoral autoimmunity in women with a shorter AR exon 1 CAG repeat length suggest a role for genetically determined sensitivity to androgens as a modulator of autoimmune processes. PMID:24711544

  2. MADS+: discovery of differential splicing events from Affymetrix exon junction array data

    PubMed Central

    Shen, Shihao; Warzecha, Claude C.; Carstens, Russ P.; Xing, Yi

    2010-01-01

    Motivation: The Affymetrix Human Exon Junction Array is a newly designed high-density exon-sensitive microarray for global analysis of alternative splicing. Contrary to the Affymetrix exon 1.0 array, which only contains four probes per exon and no probes for exon–exon junctions, this new junction array averages eight probes per probeset targeting all exons and exon–exon junctions observed in the human mRNA/EST transcripts, representing a significant increase in the probe density for alternative splicing events. Here, we present MADS+, a computational pipeline to detect differential splicing events from the Affymetrix exon junction array data. For each alternative splicing event, MADS+ evaluates the signals of probes targeting competing transcript isoforms to identify exons or splice sites with different levels of transcript inclusion between two sample groups. MADS+ is used routinely in our analysis of Affymetrix exon junction arrays and has a high accuracy in detecting differential splicing events. For example, in a study of the novel epithelial-specific splicing regulator ESRP1, MADS+ detects hundreds of exons whose inclusion levels are dependent on ESRP1, with a RT-PCR validation rate of 88.5% (153 validated out of 173 tested). Availability: MADS+ scripts, documentations and annotation files are available at http://www.medicine.uiowa.edu/Labs/Xing/MADSplus/. Contact: yi-xing@uiowa.edu PMID:19933160

  3. Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice.

    PubMed

    Lee, Eun-Joo; Kim, Ah-Young; Lee, Eun-Mi; Lee, Myeong-Mi; Min, Chang-Woo; Kang, Kyung-Ku; Park, Jin-Kyu; Hwang, Meeyul; Kwon, Soon-Hak; Tremblay, Jacques P; Jeong, Kyu-Shik

    2014-08-01

    Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor-beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.

  4. Intron-exon organization of the gene for the multifunctional animal fatty acid synthase.

    PubMed Central

    Amy, C M; Williams-Ahlf, B; Naggert, J; Smith, S

    1992-01-01

    The complete intron-exon organization of the gene encoding a multifunctional mammalian fatty acid synthase has been elucidated, and specific exons have been assigned to coding sequences for the component domains of the protein. The rat gene is interrupted by 42 introns and the sequences bordering the splice-site junctions universally follow the GT/AG rule. However, of the 41 introns that interrupt the coding region of the gene, 23 split the reading frame in phase I, 14 split the reading frame in phase 0, and only 4 split the reading frame in phase II. Remarkably, 46% of the introns interrupt codons for glycine. With only one exception, boundaries between the constituent enzymes of the multifunctional polypeptide coincide with the location of introns in the gene. The significance of the predominance of phase I introns, the almost uniformly short length of the 42 introns and the overall small size of the gene, is discussed in relation to the evolution of multifunctional proteins. Images PMID:1736293

  5. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

    PubMed Central

    Kuraoka, Mutsuki; Lee, Joshua J.A.; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  6. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    PubMed

    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  7. Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.

    PubMed

    Nussenzveig, Roberto H; Pham, Ha T; Perkins, Sherrie L; Prchal, Josef T; Agarwal, Archana M; Salama, Mohamed E

    2016-01-01

    The frequency of co-existing JAK2(V617F)/MPL and JAK2(V617F)/JAK2 exon-12 mutations has not been previously investigated in MPNs. Poor survival was reported in primary myelofibrosis with low JAK2(V617F) allelic burden. However, mutational status of JAK2 exon-12 or MPL were not reported in these patients. This study developed a cost-effective multiplex high resolution melt assay that screens for mutations in JAK2 gene exons-12 and -14 ((V617F)) and MPL gene exon-10. Co-existing mutations with JAK2(V617F) were detected in 2.9% (6/208; two JAK2 exon-12 and four MPL exon-10) patient specimens with known JAK2(V617F) (allelic-burden range: 0.1-96.8%). Co-existing mutations were detected in specimens with < 12% JAK2(V617F) allelic burden. Current WHO guidelines do not recommend further testing once JAK2(V617F) mutation is detected in MPNs. The findings, however, indicate that quantification of JAK2(V617F) allele burden may be clinically relevant in MPNs and in those with low allelic burden additional testing for JAK2 exon-12 and MPL exon-10 mutation should be pursued.

  8. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin

    SciTech Connect

    Thanh, L.T.; Man, Nguyen Thi; Morris, G.E.

    1995-08-28

    We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immunohistochemistry or Western blotting with these {open_quotes}exon-specific{close_quotes} mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying {open_quotes}revertant{close_quotes} fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene. 27 refs., 7 figs., 3 tabs.

  9. A distal enhancer and an ultraconserved exon are derived from a novel retroposon.

    PubMed

    Bejerano, Gill; Lowe, Craig B; Ahituv, Nadav; King, Bryan; Siepel, Adam; Salama, Sofie R; Rubin, Edward M; Kent, W James; Haussler, David

    2006-05-01

    Hundreds of highly conserved distal cis-regulatory elements have been characterized so far in vertebrate genomes. Many thousands more are predicted on the basis of comparative genomics. However, in stark contrast to the genes that they regulate, in invertebrates virtually none of these regions can be traced by using sequence similarity, leaving their evolutionary origins obscure. Here we show that a class of conserved, primarily non-coding regions in tetrapods originated from a previously unknown short interspersed repetitive element (SINE) retroposon family that was active in the Sarcopterygii (lobe-finned fishes and terrestrial vertebrates) in the Silurian period at least 410 million years ago (ref. 4), and seems to be recently active in the 'living fossil' Indonesian coelacanth, Latimeria menadoensis. Using a mouse enhancer assay we show that one copy, 0.5 million bases from the neuro-developmental gene ISL1, is an enhancer that recapitulates multiple aspects of Isl1 expression patterns. Several other copies represent new, possibly regulatory, alternatively spliced exons in the middle of pre-existing Sarcopterygian genes. One of these, a more than 200-base-pair ultraconserved region, 100% identical in mammals, and 80% identical to the coelacanth SINE, contains a 31-amino-acid-residue alternatively spliced exon of the messenger RNA processing gene PCBP2 (ref. 6). These add to a growing list of examples in which relics of transposable elements have acquired a function that serves their host, a process termed 'exaptation', and provide an origin for at least some of the many highly conserved vertebrate-specific genomic sequences.

  10. Sequence of the dog immunoglobulin alpha and epsilon constant region genes

    SciTech Connect

    Patel, M.; Selinger, D.; Mark, G.E.; Hollis, G.F.; Hickey, G.J.

    1995-03-01

    The immunoglobulin alpha (IGHAC) and epsilon (IGHEC) germline constant region genes were isolated from a dog liver genomic DNA library. Sequence analysis indicates that the dog IGHEC gene is encoded by four exons spread out over 1.7 kilobases (kb). The IGHAC sequence encompasses 1.5 kb and includes all three constant region coding exons. The complete exon/intron sequence of these genes is described. 28 refs., 2 figs., 2 tabs.

  11. Transposable element insertions respecify alternative exon splicing in three Drosophila myosin heavy chain mutants.

    PubMed Central

    Davis, M B; Dietz, J; Standiford, D M; Emerson, C P

    1998-01-01

    Insertions of transposable elements into the myosin heavy chain (Mhc) locus disrupt the regulation of alternative pre-mRNA splicing for multi-alternative exons in the Mhc2, Mhc3, and Mhc4 mutants in Drosophila. Sequence and expression analyses show that each inserted element introduces a strong polyadenylation signal that defines novel terminal exons, which are then differentially recognized by the alternative splicing apparatus. Mhc2 and Mhc4 have insertion elements located within intron 7c and exon 9a, respectively, and each expresses a single truncated transcript that contains an aberrant terminal exon defined by the poly(A) signal of the inserted element and the 3' acceptor of the upstream common exon. In Mhc3, a poly(A) signal inserted into Mhc intron 7d defines terminal exons using either the upstream 3' acceptor of common exon 6 or the 7d acceptor, leading to the expression of 4.1- and 1.7-kb transcripts, respectively. Acceptor selection is regulated in Mhc3 transcripts, where the 3' acceptor of common Mhc exon 6 is preferentially selected in larvae, whereas the alternative exon 7d acceptor is favored in adults. These results reflect the adult-specific use of exon 7d and suggest that the normal exon 7 alternative splicing mechanism continues to influence the selection of exon 7d in Mhc3 transcripts. Overall, transposable element-induced disruptions in alternative processing demonstrate a role for the nonconsensus 3' acceptors in Mhc exons 7 and 9 alternative splicing regulation. PMID:9799262

  12. Ternary europium mesoporous polymeric hybrid materials Eu({beta}-diketonate){sub 3}pvpd-SBA-15(16): host-guest construction, characterization and photoluminescence

    SciTech Connect

    Gu Yanjing; Yan Bing; Li Yanyan

    2012-06-15

    Novel organic-inorganic mesoporous luminescent polymeric hybrid materials containing europium(III) complexes incorporated to mesoporous silica SBA-15/SBA-16 have been prepared by simple physical doping (impregnation) methods, followed by the addition polymerization reaction of the monomer 4-vinylpyridine (vpd) extending along the mesoporous channels. The precursor europium(III) complexes are synthesized by {beta}-diketonate ({beta}-diketonate=2-thenoyltrifluoroacetonate (tta), hexafluoroacetylacetonate (hfac), trifluoroacetylacetonate (taa)) and monomer 4-vinylpyridine (vpd) coordinated to Eu{sup 3+}, and SBA-15/SBA-16 are obtained via a sol-gel process. After the physical doping and the polymerization reaction, the final ternary materials Eu({beta}-diketonate){sub 3}pvpd-SBA-15/Eu({beta}-diketonate){sub 3}pvpd-SBA-16 ({beta}-diketonate=tta, hfac, taa) are received. The physical properties and espeically the photoluminescence of these hybrids are characterized, and the XRD and BET results reveal that all of these hybrid materials have uniformity in the mesostructure. The detailed luminescence investigation on all the materials show that Eu(tta){sub 3}pvpd-SBA-16 have the highest luminescence intensity and the materials with taa ligands have longer lifetimes. - Grapical abstract: Luminescent mesoporous polymeric hybrid materials containing europium complexes hydrogen bonding to silica SBA-15/SBA-16 followed by the addition polymerization reaction of 4-vinylpyridine (vpd) extending along the mesoporous channels. Highlights: Black-Right-Pointing-Pointer Functional mesoporous with simple impregnation method. Black-Right-Pointing-Pointer New lanthanide mesoporous hybrids with polymer ligands. Black-Right-Pointing-Pointer Luminescence in visible region.

  13. Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by “exon by exon” PCR mediated SSCP analysis

    PubMed Central

    Braggio, E; Bonvicino, C R; Vargas, F R; Ferman, S; Eisenberg, A L A; Seuánez, H N

    2004-01-01

    Aims: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma. Methods: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined “exon by exon” polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing. Results: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron–exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506–5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393–572 and 646–772), whereas five were of both types—in CGA codons within E1A binding domains. Conclusions: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling. PMID:15166261

  14. ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment

    PubMed Central

    Chowdhury, Asif H.; Hasson, Dan; Dyer, Michael A.

    2016-01-01

    ABSTRACT ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3′ exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3′ exonic regions encode the zinc finger motifs, which can range from 1–40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3′ exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3′ exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3′ exons of ZNFs to maintain their genomic stability through preservation of H3K9me3. PMID:27029610

  15. Diversity in exon 5 of HLA-C(∗)04:01:01G is significant in anthropological studies.

    PubMed

    Dunn, Paul P J; Lamb, Gareth; Selwyn, Caroline; Compton, Jillian; Yang, Edward; Maiers, Martin; Fernandez-Vina, Marcelo

    2016-05-01

    Polymorphisms in Human Leukocyte Antigen (HLA) class I genes are generally considered to be relevant only if they reside in exons 2 and 3 or if they affect the expression of the allele. HLA-C(∗)04:82 differs from the common HLA-C(∗)04:01:01 by having a 9 nucleotide, or 3 amino acid duplication, in exon 5. Having observed HLA-C(∗)04:82 in a New Zealand Maori stem cell patient, we have attempted to examine the prevalence of this allele in different ethnicities. Although our studies are in a limited number of patients and donors, they have revealed that, in the Pacific region, HLA-C(∗)04:82 appears to be the most common allele of the HLA-C(∗)04:01:01G group of alleles, notably in Filippinos and in Maori/Polynesians. In these populations this allele has characteristic HLA-ABCDRB1 haplotypes. Thus, our studies have shown that polymorphisms outside of the clinically important exons can be considered to be relevant in anthropological studies. PMID:27018403

  16. A New Chromosome X Exon-Specific Microarray Platform for Screening of Patients with X-Linked Disorders

    PubMed Central

    Bashiardes, Stavros; Kousoulidou, Ludmila; van Bokhoven, Hans; Ropers, Hans-Hilger; Chelly, Jamel; Moraine, Claude; de Brouwer, Arjan P.M.; Van Esch, Hilde; Froyen, Guy; Patsalis, Philippos C.

    2009-01-01

    Recent studies and advances in high-density oligonucleotide arrays have shown that microdeletions and microduplications occur at a high frequency in the human genome, causing various genetic conditions including mental retardation. Thus far little is known about the pathways leading to this disease, and implementation of microarrays is hampered by their increasing cost and complexity, underlining the need for new diagnostic tools. The aim of this study was to introduce a new targeted platform called “chromosome X exon-specific array” and to apply this new platform to screening of 20 families (including one blind positive control) with suspected X-linked mental retardation, to identify new causative X-linked mental retardation genes. The new microarray contains of 21,939 oligonucleotides covering 92.9% of all exons of all genes on chromosome X. Patient screening resulted in successful identification of the blind positive control included in the sample of 20 families, and one of the remaining 19 families was found to carry a 1.78-kilobase deletion involving all exons of pseudogene BRAF2. The BRAF2 deletion segregated in the family and was not found in 200 normal male samples, and no copy number variations are reported in this region. Further studies and focused investigation of X-linked disorders have the potential to reveal the molecular basis of human genetic pathological conditions that are caused by copy-number changes in chromosome X genes. PMID:19779134

  17. A new chromosome x exon-specific microarray platform for screening of patients with X-linked disorders.

    PubMed

    Bashiardes, Stavros; Kousoulidou, Ludmila; van Bokhoven, Hans; Ropers, Hans-Hilger; Chelly, Jamel; Moraine, Claude; de Brouwer, Arjan P M; Van Esch, Hilde; Froyen, Guy; Patsalis, Philippos C

    2009-11-01

    Recent studies and advances in high-density oligonucleotide arrays have shown that microdeletions and microduplications occur at a high frequency in the human genome, causing various genetic conditions including mental retardation. Thus far little is known about the pathways leading to this disease, and implementation of microarrays is hampered by their increasing cost and complexity, underlining the need for new diagnostic tools. The aim of this study was to introduce a new targeted platform called "chromosome X exon-specific array" and to apply this new platform to screening of 20 families (including one blind positive control) with suspected X-linked mental retardation, to identify new causative X-linked mental retardation genes. The new microarray contains of 21,939 oligonucleotides covering 92.9% of all exons of all genes on chromosome X. Patient screening resulted in successful identification of the blind positive control included in the sample of 20 families, and one of the remaining 19 families was found to carry a 1.78-kilobase deletion involving all exons of pseudogene BRAF2. The BRAF2 deletion segregated in the family and was not found in 200 normal male samples, and no copy number variations are reported in this region. Further studies and focused investigation of X-linked disorders have the potential to reveal the molecular basis of human genetic pathological conditions that are caused by copy-number changes in chromosome X genes.

  18. Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function.

    PubMed

    Oda, Hirotsugu; Sato, Tatsuhiro; Kunishima, Shinji; Nakagawa, Kenji; Izawa, Kazushi; Hiejima, Eitaro; Kawai, Tomoki; Yasumi, Takahiro; Doi, Hiraku; Katamura, Kenji; Numabe, Hironao; Okamoto, Shinya; Nakase, Hiroshi; Hijikata, Atsushi; Ohara, Osamu; Suzuki, Hidenori; Morisaki, Hiroko; Morisaki, Takayuki; Nunoi, Hiroyuki; Hattori, Seisuke; Nishikomori, Ryuta; Heike, Toshio

    2016-03-01

    Loss-of-function mutations in filamin A (FLNA) cause an X-linked dominant disorder with multiple organ involvement. Affected females present with periventricular nodular heterotopia (PVNH), cardiovascular complications, thrombocytopenia and Ehlers-Danlos syndrome. These mutations are typically lethal to males, and rare male survivors suffer from failure to thrive, PVNH, and severe cardiovascular and gastrointestinal complications. Here we report two surviving male siblings with a loss-of-function mutation in FLNA. They presented with multiple complications, including valvulopathy, intestinal malrotation and chronic intestinal pseudo-obstruction (CIPO). However, these siblings had atypical clinical courses, such as a lack of PVNH and a spontaneous improvement of CIPO. Trio-based whole-exome sequencing revealed a 4-bp deletion in exon 40 that was predicted to cause a lethal premature protein truncation. However, molecular investigations revealed that the mutation induced in-frame skipping of the mutated exon, which led to the translation of a mutant FLNA missing an internal region of 41 amino acids. Functional analyses of the mutant protein suggested that its binding affinity to integrin, as well as its capacity to induce focal adhesions, were comparable to those of the wild-type protein. These results suggested that exon skipping of FLNA partially restored its protein function, which could contribute to amelioration of the siblings' clinical courses. This study expands the diversity of the phenotypes associated with loss-of-function mutations in FLNA. PMID:26059841

  19. Direct evidence for calcineurin binding to the exon-7 loop of the sodium-bicarbonate cotransporter NBCn1.

    PubMed

    Gill, Harindarpal S; Roush, Eric D; Dutcher, Lauren; Patel, Samir

    2014-01-01

    The NaHCO3 cotransporter NBCn1 plays a role in neutralizing intracellular acid loads at the basolateral membrane in cells of the medullary thick ascending limb (mTAL). Calcineurin inhibitors (Cn-Is) are known to both downregulate NBCn1 expression in the distal nephron and cause renal tubular acidosis (RTA), a risk factor for nephrocalcinosis and nephrolithiasis. In this report, we provide a new perspective on concurrent studies of NBCn1 in various tissues by using cell-free binding assays to investigate the interaction of NBCn1 with the calcineurin (Cn) isoform PPP3CA. Surface plasmon resonance (SPR) analyses show that the protein domain Exon 7 (translated from cassette II of NBCn1) binds Cn with an equilibrium dissociation constant (KD) of 30 +/- 15 nm. Linked-reaction tests suggest that the binding involves a conformational change. Nested PCR reactions were used to show that NBCn1-Exon 7 splice variants with alternative N-termini regions are expressed in the kidney, as well as other tissues. Additionally, we discuss NBCn1-Exon 7 implication in acid-base balance and calcium crystallization in the kidney. PMID:25076853

  20. Novel methodologies for spectral classification of exon and intron sequences

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  1. Characterization of two exon-skipping mutations (3120G{r_arrow}A, 3600G{r_arrow}A) in the CFTR gene

    SciTech Connect

    Zielendki, J.; Markiewicz, D.; Ainsworth, P.J.

    1994-09-01

    Many different types of mutations have been identified in the CFTR gene in patients with cystic fibrosis. Due to the large size of the gene (230 kb), CF mutations have been primarily detected by genomic DNA analysis. While some of the sequence alterations, such as nonsense and frameshift mutations, provide immediate clues to possible molecular consequence, others such as missense mutations are less apparent in their involvement in the disease. In our systematic scanning of the entire coding regions of the CFTR gene for a group of CF patients carrying unknown mutations, two different G to A substitutions located at the last nucleotide position of an exon were identified in two patients. The first one, 3120G{r_arrow}A, is located in exon 16 and the other one, 3600G{r_arrow}A, in exon 18 of the CFTR gene. Both of them are also located at the third position of the corresponding amino acid codon (CAG and TTC, respectively). As a result, the changes would not affect the encoded amino acids (Glu and Leu, respectively). To demonstrate that these are in fact pathologic mutations, we have investigated the CFTR transcripts in these two patients. The results of RT-PCR analysis revealed that aberrant splicing occurred in both cases: transcripts missing exon 16 and 18 were present in the 2 patients, respectively. No normal product was detectable from the 3120G{r_arrow}A and 3600G{r_arrow}A alleles, suggesting that the normal-sized products were exclusively derived from the {triangle}F508 mutant alleles in both of these patients. Hence, we conclude that both 3120G{r_arrow}A and 3600G{r_arrow}A mutations cause exon-skipping leading to premature termination and truncation of CFTR and that the altered G residue in each of these exons is probably part of the splice donor sequence important for efficient mRNA splicing.

  2. IUGR increases chromatin-remodeling factor Brg1 expression and binding to GR exon 1.7 promoter in newborn male rat hippocampus.

    PubMed

    Ke, Xingrao; McKnight, Robert A; Gracey Maniar, Lia E; Sun, Ying; Callaway, Christopher W; Majnik, Amber; Lane, Robert H; Cohen, Susan S

    2015-07-15

    Intrauterine growth restriction (IUGR) increases the risk for neurodevelopment delay and neuroendocrine reprogramming in both humans and rats. Neuroendocrine reprogramming involves the glucocorticoid receptor (GR) gene that is epigenetically regulated in the hippocampus. Using a well-characterized rodent model, we have previously shown that IUGR increases GR exon 1.7 mRNA variant and total GR expressions in male rat pup hippocampus. Epigenetic regulation of GR transcription may involve chromatin remodeling of the GR gene. A key chromatin remodeler is Brahma-related gene-1(Brg1), a member of the ATP-dependent SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Brg1 regulates gene expression by affecting nucleosome repositioning and recruiting transcriptional components to target promoters. We hypothesized that IUGR would increase hippocampal Brg1 expression and binding to GR exon 1.7 promoter, as well as alter nucleosome positioning over GR promoters in newborn male pups. Further, we hypothesized that IUGR would lead to accumulation of specificity protein 1 (Sp1) and RNA pol II at GR exon 1.7 promoter. Indeed, we found that IUGR increased Brg1 expression and binding to GR exon 1.7 promoter. We also found that increased Brg1 binding to GR exon 1.7 promoter was associated with accumulation of Sp1 and RNA pol II carboxy terminal domain pSer-5 (a marker of active transcription). Furthermore, the transcription start site of GR exon 1.7 was located within a nucleosome-depleted region. We speculate that changes in hippocampal Brg1 expression mediate GR expression and subsequently trigger neuroendocrine reprogramming in male IUGR rats. PMID:25972460

  3. Dipole angular entropy techniques for intron-exon segregation in DNA

    NASA Astrophysics Data System (ADS)

    Subramanian, Nithya; Bose, R.

    2012-04-01

    We propose techniques for computing the angular entropies of DNA sequences, based on the orientations of the dipole moments of the nucleotide bases. The angles of the dipole moment vectors of the bases are used to compute the dipole angular entropy and the Fourier harmonics of the angles are used to compute the dipole angular spectral entropy for a given sequence. We also show that the coding (exons) and noncoding (introns) regions of the DNA can be segregated based on their dipole angular entropies and dipole angular spectral entropies. Segregation using these techniques is found to be computationally faster and more accurate than the previously reported methods. The proposed techniques can also be improvised to use the magnitude of the dipole moments of the bases in addition to the angles.

  4. Power of deep, all-exon resequencing for discovery of human trait genes

    PubMed Central

    Kryukov, Gregory V.; Shpunt, Alexander; Stamatoyannopoulos, John A.; Sunyaev, Shamil R.

    2009-01-01

    The ability to sequence cost-effectively all of the coding regions of a given individual genome is rapidly approaching, with the potential for whole-genome resequencing not far behind. Initiatives are currently underway to phenotype hundreds of thousands of individuals for major human traits. Here, we determine the power for de novo discovery of genes related to human traits by resequencing all human exons in a clinical population. We analyze the potential of the gene discovery strategy that combines multiple rare variants from the same gene and treats genes, rather than individual alleles, as the units for the association test. By using computer simulations based on deep resequencing data for the European population, we show that genes meaningfully affecting a human trait can be identified in an unbiased fashion, although large sample sizes would be required to achieve substantial power. PMID:19202052

  5. TALE-directed local modulation of H3K9 methylation shapes exon recognition.

    PubMed

    Bieberstein, Nicole I; Kozáková, Eva; Huranová, Martina; Thakur, Prasoon K; Krchňáková, Zuzana; Krausová, Michaela; Carrillo Oesterreich, Fernando; Staněk, David

    2016-07-21

    In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

  6. The complete local genotype–phenotype landscape for the alternative splicing of a human exon

    PubMed Central

    Julien, Philippe; Miñana, Belén; Baeza-Centurion, Pablo; Valcárcel, Juan; Lehner, Ben

    2016-01-01

    The properties of genotype–phenotype landscapes are crucial for understanding evolution but are not characterized for most traits. Here, we present a >95% complete local landscape for a defined molecular function—the alternative splicing of a human exon (FAS/CD95 exon 6, involved in the control of apoptosis). The landscape provides important mechanistic insights, revealing that regulatory information is dispersed throughout nearly every nucleotide in an exon, that the exon is more robust to the effects of mutations than its immediate neighbours in genotype space, and that high mutation sensitivity (evolvability) will drive the rapid divergence of alternative splicing between species unless it is constrained by selection. Moreover, the extensive epistasis in the landscape predicts that exonic regulatory sequences may diverge between species even when exon inclusion levels are functionally important and conserved by selection. PMID:27161764

  7. TALE-directed local modulation of H3K9 methylation shapes exon recognition

    PubMed Central

    Bieberstein, Nicole I.; Kozáková, Eva; Huranová, Martina; Thakur, Prasoon K.; Krchňáková, Zuzana; Krausová, Michaela; Oesterreich, Fernando Carrillo; Staněk, David

    2016-01-01

    In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons. PMID:27439481

  8. Hypomethylation of the interphotoreceptor retinoid-binding protein (IRBP) promotor and first exon is linked to expression of the gene.

    PubMed Central

    Albini, A; Toffenetti, J; Zhu, Z; Chader, G J; Noonan, D M

    1990-01-01

    The interphotoreceptor retinoid-binding protein (IRBP) is limited in expression to retinal photoreceptor cells and a subset of pinealocytes. We have obtained a genomic clone containing the entire coding region and 7 kb of 5' flanking sequence. As a first step in studying IRBP gene regulation we have examined the CpG methylation patterns of the entire IRBP gene in expressing and non-expressing human cells. This has been done by isolation of high molecular weight DNA from Y-79 cells grown in suspension or attached to poly-D-lysine, which synthesize IRBP at different levels, and from human lymphocytes, which were shown by northern analysis to lack IRBP message. The DNA was digested by either Hpa II, Msp I, or Hha I. Southern blots were prepared with these digests and hybridized with probes made from fragments covering the complete genomic clone. Probes from the first exon, the introns and the 3' end gave banding patterns which showed no differences between the expressing cells and the lymphocytes. A probe from the very 5' end did not give a clear banding pattern, probably due to the presence of repetitive elements in the probe. However, a Hind III probe covering the 5' flanking 3 kb and the beginning of the first exon hybridized with a 1.8 kb band in Hpa II digests of Y-79 cells which was not present in Hpa II digests of lymphocyte DNA. In addition, a 2.1-2.3 kb Hha I band was found only in the Y-79 DNA digests. Sequence analysis of the promoter region indicated that these bands were due to hypomethylation of sites within a CpG rich island from -1578 to -1108 in the promoter and hypomethylation of sites in the beginning of the first exon. A Hha I site between the CpG island and the first exon was not hypomethylated in the expressing Y-79 cells. We propose that hypomethylation of the CpG rich island of the IRBP promoter and the first exon is linked to the expression of this gene. Images PMID:2402443

  9. The exon quantification pipeline (EQP): a comprehensive approach to the quantification of gene, exon and junction expression from RNA-seq data.

    PubMed

    Schuierer, Sven; Roma, Guglielmo

    2016-09-19

    The quantification of transcriptomic features is the basis of the analysis of RNA-seq data. We present an integrated alignment workflow and a simple counting-based approach to derive estimates for gene, exon and exon-exon junction expression. In contrast to previous counting-based approaches, EQP takes into account only reads whose alignment pattern agrees with the splicing pattern of the features of interest. This leads to improved gene expression estimates as well as to the generation of exon counts that allow disambiguating reads between overlapping exons. Unlike other methods that quantify skipped introns, EQP offers a novel way to compute junction counts based on the agreement of the read alignments with the exons on both sides of the junction, thus providing a uniformly derived set of counts. We evaluated the performance of EQP on both simulated and real Illumina RNA-seq data and compared it with other quantification tools. Our results suggest that EQP provides superior gene expression estimates and we illustrate the advantages of EQP's exon and junction counts. The provision of uniformly derived high-quality counts makes EQP an ideal quantification tool for differential expression and differential splicing studies. EQP is freely available for download at https://github.com/Novartis/EQP-cluster.

  10. Non-hominid TP63 lacks retroviral LTRs but contains a novel conserved upstream exon.

    PubMed

    Beyer, Ulrike; Dobbelstein, Matthias

    2011-06-15

    We have recently identified novel isoforms of human p63, with specific expression in testicular germ cells. The synthesis of these p63 mRNA species is driven by the long terminal repeat (LTR) of the endogenous retrovirus ERV9. This LTR was inserted upstream of the previously known TP63 exons roughly 15 million years ago, leading to the expression of novel exons and the synthesis of germline-specific transactivating p63 (GTAp63) isoforms in humans and great apes (Beyer et al. Proc Natl Acad Sci USA 2011; 108:3624-9). However, this study did not reveal whether similar upstream exons can also be found in the TP63 genes of non-hominid animals. Here we performed rapid amplification of cDNA ends (RACE) to identify a novel upstream exon of murine TP63, located in the 5' position from the previously described start of transcription. This exon, termed "exon U3" in our previous publication, is conserved within a broad range of mammalian species, including hominids. However, in contrast to the human TP63 gene structure, the murine exon U3 represented the most upstream transcribed sequence of TP63. Murine exon U3 is then alternatively spliced to acceptor sites within exon 1 or upstream of exon 2, resulting in two different available translational start sites. p63 mRNAs comprising exon U3 are detectable in various tissues, with no particular preference for testicular cells. Thus, whereas the retroviral LTR in hominid species results in strictly germline-associated p63 isoforms, the upstream exon in non-hominids fails to confer this tissue specificity. This notion strongly supports the concept that the synthesis of a testis-specific p63 isoform is a recently acquired, unique feature of humans and great apes.

  11. Assembling genes from predicted exons in linear time with dynamic programming.

    PubMed

    Guigó, R

    1998-01-01

    In a number of programs for gene structure prediction in higher eukaryotic genomic sequences, exon prediction is decoupled from gene assembly: a large pool of candidate exons is predicted and scored from features located in the query DNA sequence, and candidate genes are assembled from such a pool as sequences of nonoverlapping frame-compatible exons. Genes are scored as a function of the scores of the assembled exons, and the highest scoring candidate gene is assumed to be the most likely gene encoded by the query DNA sequence. Considering additive gene scoring functions, currently available algorithms to determine such a highest scoring candidate gene run in time proportional to the square of the number of predicted exons. Here, we present an algorithm whose running time grows only linearly with the size of the set of predicted exons. Polynomial algorithms rely on the fact that, while scanning the set of predicted exons, the highest scoring gene ending in a given exon can be obtained by appending the exon to the highest scoring among the highest scoring genes ending at each compatible preceding exon. The algorithm here relies on the simple fact that such highest scoring gene can be stored and updated. This requires scanning the set of predicted exons simultaneously by increasing acceptor and donor position. On the other hand, the algorithm described here does not assume an underlying gene structure model. Indeed, the definition of valid gene structures is externally defined in the so-called Gene Model. The Gene Model specifies simply which gene features are allowed immediately upstream which other gene features in valid gene structures. This allows for great flexibility in formulating the gene identification problem. In particular it allows for multiple-gene two-strand predictions and for considering gene features other than coding exons (such as promoter elements) in valid gene structures.

  12. Exon amendment: threat to AIDS prevention and activism?

    PubMed

    Mirken, B

    1995-07-21

    The controversial Communications Decency Act of 1995, frequently referred to as the Exon amendment after its author, Sen. James Exon of Nebraska, may prove to be a threat to AIDS prevention and activism. The measure, an amendment to the Telecommunications Competition and Deregulation Act of 1995, passed the Senate and may soon be considered by the House of Representatives. The amendment makes it a crime to make or make available any obscene or indecent communication in any form to a person under eighteen years of age. The measure also criminalizes the owners or operators of any telecommunications facility used for such purposes. Of concern is how much AIDS-related material available online might be considered indecent. Currently, there are a number of AIDS bulletin boards and Internet groups that have HIV/AIDS discussions containing frank, graphic dialogue of the HIV risks involved in various sexual acts. Whether they are obscene or indecent will likely be decided by the courts. Although not yet a major vehicle for AIDS prevention information, experts worry what the effects will be if online information is restricted.

  13. Genomic V exons from whole genome shotgun data in reptiles.

    PubMed

    Olivieri, D N; von Haeften, B; Sánchez-Espinel, C; Faro, J; Gambón-Deza, F

    2014-08-01

    Reptiles and mammals diverged over 300 million years ago, creating two parallel evolutionary lineages amongst terrestrial vertebrates. In reptiles, two main evolutionary lines emerged: one gave rise to Squamata, while the other gave rise to Testudines, Crocodylia, and Aves. In this study, we determined the genomic variable (V) exons from whole genome shotgun sequencing (WGS) data in reptiles corresponding to the three main immunoglobulin (IG) loci and the four main T cell receptor (TR) loci. We show that Squamata lack the TRG and TRD genes, and snakes lack the IGKV genes. In representative species of Testudines and Crocodylia, the seven major IG and TR loci are maintained. As in mammals, genes of the IG loci can be grouped into well-defined IMGT clans through a multi-species phylogenetic analysis. We show that the reptilian IGHV and IGLV genes are distributed amongst the established mammalian clans, while their IGKV genes are found within a single clan, nearly exclusive from the mammalian sequences. The reptilian and mammalian TRAV genes cluster into six common evolutionary clades (since IMGT clans have not been defined for TR). In contrast, the reptilian TRBV genes cluster into three clades, which have few mammalian members. In this locus, the V exon sequences from mammals appear to have undergone different evolutionary diversification processes that occurred outside these shared reptilian clans. These sequences can be obtained in a freely available public repository (http://vgenerepertoire.org).

  14. Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta.

    PubMed

    Poulter, James A; Murillo, Gina; Brookes, Steven J; Smith, Claire E L; Parry, David A; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

    2014-10-15

    Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid.

  15. JuncDB: an exon–exon junction database

    PubMed Central

    Chorev, Michal; Guy, Lotem; Carmel, Liran

    2016-01-01

    Intron positions upon the mRNA transcript are sometimes remarkably conserved even across distantly related eukaryotic species. This has made the comparison of intron–exon architectures across orthologous transcripts a very useful tool for studying various evolutionary processes. Moreover, the wide range of functions associated with introns may confer biological meaning to evolutionary changes in gene architectures. Yet, there is currently no database that offers such comparative information. Here, we present JuncDB (http://juncdb.carmelab.huji.ac.il/), an exon–exon junction database dedicated to the comparison of architectures between orthologous transcripts. It covers nearly 40 000 sets of orthologous transcripts spanning 88 eukaryotic species. JuncDB offers a user-friendly interface, access to detailed information, instructive graphical displays of the comparative data and easy ways to download data to a local computer. In addition, JuncDB allows the analysis to be carried out either on specific genes, or at a genome-wide level for any selected group of species. PMID:26519469

  16. A Relaxed Active Site After Exon Ligation by the Group I Intron

    SciTech Connect

    Lipchock,S.; Strobel, S.

    2008-01-01

    During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.

  17. SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene.

    PubMed

    Moon, Heegyum; Cho, Sunghee; Loh, Tiing Jen; Oh, Hyun Kyung; Jang, Ha Na; Zhou, Jianhua; Kwon, Young-Soo; Liao, D Joshua; Jun, Youngsoo; Eom, Soohyun; Ghigna, Claudia; Biamonti, Giuseppe; Green, Michael R; Zheng, Xuexiu; Shen, Haihong

    2014-11-01

    The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.

  18. Coding exon-structure aware realigner (CESAR) utilizes genome alignments for accurate comparative gene annotation.

    PubMed

    Sharma, Virag; Elghafari, Anas; Hiller, Michael

    2016-06-20

    Identifying coding genes is an essential step in genome annotation. Here, we utilize existing whole genome alignments to detect conserved coding exons and then map gene annotations from one genome to many aligned genomes. We show that genome alignments contain thousands of spurious frameshifts and splice site mutations in exons that are truly conserved. To overcome these limitations, we have developed CESAR (Coding Exon-Structure Aware Realigner) that realigns coding exons, while considering reading frame and splice sites of each exon. CESAR effectively avoids spurious frameshifts in conserved genes and detects 91% of shifted splice sites. This results in the identification of thousands of additional conserved exons and 99% of the exons that lack inactivating mutations match real exons. Finally, to demonstrate the potential of using CESAR for comparative gene annotation, we applied it to 188 788 exons of 19 865 human genes to annotate human genes in 99 other vertebrates. These comparative gene annotations are available as a resource (http://bds.mpi-cbg.de/hillerlab/CESAR/). CESAR (https://github.com/hillerlab/CESAR/) can readily be applied to other alignments to accurately annotate coding genes in many other vertebrate and invertebrate genomes. PMID:27016733

  19. Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production.

    PubMed Central

    Mineo, I; Holmes, E W

    1991-01-01

    Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene. Images PMID:1922051

  20. SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene

    PubMed Central

    Moon, Heegyum; Cho, Sunghee; Loh, Tiing Jen; Oh, Hyun Kyung; Jang, Ha Na; Zhou, Jianhua; Kwon, Young-Soo; Liao, D. Joshua; Jun, Youngsoo; Eom, Soohyun; Ghigna, Claudia; Biamonti, Giuseppe; Green, Michael R; Zheng, Xuexiu; Shen, Haihong

    2015-01-01

    The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induce a decrease in the splicing of both intron 10 and 11, by contrast, overexpression of SRSF2 induce an increase in the splicing of intron 10 and 11. Through mutation analysis, we show that SRSF2 functionally target and physically interact with CGAG sequence on exon 11. In addition, we reveal that weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed. PMID:25220236

  1. Gene structure for the. alpha. 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon

    SciTech Connect

    Tikka, L.; Pihlajaniemi, T.; Henttu, P.; Prockop, D.J.; Tryggvason, K. )

    1988-10-01

    Two overlapping human genomic clones that encode a short-chain collagen, designated {alpha}1(XIII), were isolated by using recently described cDNA clones. Characterization of the cosmid clones that span {approx} 65,000 base pairs (bp) of the 3' end of the gene established several unusual features of this collagen gene. The last exon encodes solely the 3' untranslated region and it begins with a complete stop codon. The 10 adjacent exons vary in size from 27 to 87 bp and two of them are 54 bp. Therefore, the {alpha}1-chain gene of type XIII collagen has some features found in genes for fibrillar collagens but other features that are distinctly different. Previous analysis of overlapping cDNA clones and nuclease S1 mapping of mRNAs indicated one alternative splicing site causing a deletion of 36 bp from the mature mRNA. The present study showed that the 36 bp is contained within the gene as a single exon and also that the gene has a 45-bp -Gly-Xaa-Xaa- repeat coding exon not found in the cDNA clones previously characterized. Nuclease S1 mapping experiments indicated that this 45-bp exon is found in normal human skin fibroblast mRNAs. Accordingly, the data demonstrate that there is alternative splicing of at least two exons of the type {alpha}1(XIII)-chain gene.

  2. Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

    PubMed Central

    Garbarz, M; Tse, W T; Gallagher, P G; Picat, C; Lecomte, M C; Galibert, F; Dhermy, D; Forget, B G

    1991-01-01

    The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain. Images PMID:2056132

  3. Inherited human complement C5 deficiency: Nonsense mutations in exons 1 (Gln{sup 1} to Stop) and 36 (Arg{sup 1458} to Stop) and compound heterozygosity in three African-American families

    SciTech Connect

    Wang, X.; Fleischer, D.T.; Whitehead, W.T.

    1995-05-15

    Hereditary C5 deficiency has been reported in several families of different ethnic backgrounds and from different geographic regions, but the molecular genetic defect causing C5 deficiency has not been delineated in any of them. To examine the molecular basis of C5 deficiency in the African-American population, the exons and intron/exon boundaries of the C5 structural genes from three C5-deficient (C5D) African-American families were sequenced, revealing two nonsense mutations. The nonsense mutations are located in exon 1 (C{sup 84}AG to TAG) in two of the C5D families (Rhode Island and North Carolina) and in exon 36 (C{sup 4521}GA to TGA) in the third C5D family (New York). The exon 1 and 36 mutations are contained in codons that encode the first amino acid of the C5 {beta}-chain (Gln{sup 1} to Stop) and residue 1458 in the {alpha}-chain (Arg{sup 1458} to Stop), respectively. Allele-specific PCR and sequence analyses demonstrated that the exon 1 mutation is present in only one of the C5 null genes in both the Rhode Island and North Carolina families, and the exon 36 mutation is contained in only one C5 null gene in the New York family. Neither of the nonsense mutations was found in the European or Caucasian-American C5D individuals examined. Collectively, these data indicate that: (1) C5 deficiency is caused by several different molecular genetic defects, (2) C5 deficiency in the African-American population can be explained in part by two distinct nonsense mutations in exons 1 and 36, and (3) compound heterozygosity exists in all of the reported African-American C5D families. 44 refs., 5 figs., 1 tab.

  4. Elevation of BDNF exon I-specific transcripts in the frontal cortex and midbrain of rat during spontaneous morphine withdrawal is accompanied by enhanced pCreb1 occupancy at the corresponding promoter.

    PubMed

    Peregud, Danil I; Panchenko, Leonid F; Gulyaeva, Natalia V

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) is believed to play a crucial role in the mechanisms underlying opiate dependence; however, little is known about specific features and mechanisms regulating its expression in the brain under these conditions. The aim of this study was to investigate the effects of acute morphine intoxication and withdrawal from chronic intoxication on expression of BDNF exon I-, II-, IV-, VI- and IX-containing transcripts in the rat frontal cortex and midbrain. We also have studied whether alterations of BDNF exon-specific transcripts are accompanied by changes in association of well-known transcriptional regulators of BDNF gene-phosphorylated (active form) cAMP response element binding protein (pCreb1) and methyl-CpG binding protein 2 (MeCP2) with corresponding regulatory regions of the BDNF gene. Acute morphine intoxication did not affect levels of BDNF exons in brain regions, while spontaneous morphine withdrawal in dependent rats was accompanied by an elevation of the BDNF exon I-containing mRNAs both in the frontal cortex and midbrain. During spontaneous morphine withdrawal, increased associations of pCreb1 were found with promoter of exon I in the frontal cortex and promoters of exon I, IV and VI in the midbrain. The association of MeCP2 with BDNF promoters during spontaneous morphine withdrawal did not change. Thus, BDNF exon-specific transcripts are differentially expressed in brain regions during spontaneous morphine withdrawal in dependent rats and pCreb1 may be at least partially responsible for these alterations.

  5. Major histocompatibility complex class I evolution in songbirds: universal primers, rapid evolution and base compositional shifts in exon 3

    PubMed Central

    Alcaide, Miguel; Liu, Mark

    2013-01-01

    Genes of the Major Histocompatibility Complex (MHC) have become an important marker for the investigation of adaptive genetic variation in vertebrates because of their critical role in pathogen resistance. However, despite significant advances in the last few years the characterization of MHC variation in non-model species still remains a challenging task due to the redundancy and high variation of this gene complex. Here we report the utility of a single pair of primers for the cross-amplification of the third exon of MHC class I genes, which encodes the more polymorphic half of the peptide-binding region (PBR), in oscine passerines (songbirds; Aves: Passeriformes), a group especially challenging for MHC characterization due to the presence of large and complex MHC multigene families. In our survey, although the primers failed to amplify exon 3 from two suboscine passerine birds, they amplified exon 3 of multiple MHC class I genes in all 16 species of oscine songbirds tested, yielding a total of 120 sequences. The 16 songbird species belong to 14 different families, primarily within the Passerida, but also in the Corvida. Using a conservative approach based on the analysis of cloned amplicons (n = 16) from each species, we found between 3 and 10 MHC sequences per individual. Each allele repertoire was highly divergent, with the overall number of polymorphic sites per species ranging from 33 to 108 (out of 264 sites) and the average number of nucleotide differences between alleles ranging from 14.67 to 43.67. Our survey in songbirds allowed us to compare macroevolutionary dynamics of exon 3 between songbirds and non-passerine birds. We found compelling evidence of positive selection acting specifically upon peptide-binding codons across birds, and we estimate the strength of diversifying selection in songbirds to be about twice that in non-passerines. Analysis using comparative methods suggest weaker evidence for a higher GC content in the 3rd codon position of

  6. Position-dependent correlations between DNA methylation and the evolutionary rates of mammalian coding exons

    PubMed Central

    Chuang, Trees-Juen; Chen, Feng-Chi; Chen, Yen-Zho

    2012-01-01

    DNA cytosine methylation is a central epigenetic marker that is usually mutagenic and may increase the level of sequence divergence. However, methylated genes have been reported to evolve more slowly than unmethylated genes. Hence, there is a controversy on whether DNA methylation is correlated with increased or decreased protein evolutionary rates. We hypothesize that this controversy has resulted from the differential correlations between DNA methylation and the evolutionary rates of coding exons in different genic positions. To test this hypothesis, we compare human–mouse and human–macaque exonic evolutionary rates against experimentally determined single-base resolution DNA methylation data derived from multiple human cell types. We show that DNA methylation is significantly related to within-gene variations in evolutionary rates. First, DNA methylation level is more strongly correlated with C-to-T mutations at CpG dinucleotides in the first coding exons than in the internal and last exons, although it is positively correlated with the synonymous substitution rate in all exon positions. Second, for the first exons, DNA methylation level is negatively correlated with exonic expression level, but positively correlated with both nonsynonymous substitution rate and the sample specificity of DNA methylation level. For the internal and last exons, however, we observe the opposite correlations. Our results imply that DNA methylation level is differentially correlated with the biological (and evolutionary) features of coding exons in different genic positions. The first exons appear more prone to the mutagenic effects, whereas the other exons are more influenced by the regulatory effects of DNA methylation. PMID:23019368

  7. The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern.

    PubMed Central

    Elima, K; Eerola, I; Rosati, R; Metsäranta, M; Garofalo, S; Perälä, M; De Crombrugghe, B; Vuorio, E

    1993-01-01

    Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3'-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3'-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice. Images Figure 3 Figure 5 Figure 6 PMID:8424763

  8. Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

    PubMed Central

    Teigelkamp, S; Newman, A J; Beggs, J D

    1995-01-01

    Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP. Images PMID:7781612

  9. The comparison of different pre- and post-analysis filters for determination of exon-level alternative splicing events using Affymetrix arrays.

    PubMed

    Whistler, Toni; Chiang, Cheng-Feng; Lin, Jin-Mann; Lonergan, William; Reeves, William C

    2010-04-01

    Understanding the biologic significance of alternative splicing has been impeded by the difficulty in systematically identifying and validating transcript isoforms. Current exon array workflows suggest several different filtration steps to reduce the number of tests and increase the detection of alternative splicing events. In this study, we examine the effects of the suggested pre-analysis filtration by detection above background P value or signal intensity. This is followed post-analytically by restriction of exon expression to a fivefold change between groups, limiting the analysis to known alternative splicing events, or using the intersection of the results from different algorithms. Combinations of the filters are also examined. We find that none of the filtering methods reduces the number of technical false-positive calls identified by visual inspection. These include edge effects, nonresponsive probe sets, and inclusion of intronic and untranslated region probe sets into transcript annotations. Modules for filtering the exon microarray data on the basis of annotation features are needed. We propose new approaches to data filtration that would reduce the number of technical false-positives and therefore, impact the time spent performing visual inspection of the exon arrays.

  10. Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

    NASA Technical Reports Server (NTRS)

    Kretsinger, R. H.; Nakayama, S.

    1993-01-01

    In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

  11. A simple method for examination of polymorphisms of catalase exon 9: rs769217 in Hungarian microcytic anemia and beta-thalassemia patients.

    PubMed

    Nagy, Teréz; Csordás, Melinda; Kósa, Zsuzsanna; Góth, László

    2012-09-15

    Catalase decreases the high, toxic concentrations of hydrogen peroxide but it lets the physiological, low concentrations in the cells mainly for signaling purposes. Its decreased activity may contribute to development of several pathological conditions. Catalase mutations occur frequently in exon 9, these were examined with different, complicated and costly methods. The aim of the current study was to evaluate a method for screening of polymorphisms in catalase exon 9. We used the slab gel electrophoresis of PCR amplicons without denaturation and silver staining for visualization of the DNA bands. We detected extra DNA bands in the 400-800 bp region of the catalase exon 9. Their single stranded nature was proved with nucleotide sequence analyses, comparison with the standard SSCP, staining with Sybr Green II and Sybr Green I, ethidium bromide, no digestion with RFLP (BstX I), and digestion with plant nuclease. We used this method for examination of polymorphisms of catalase exon 9 in microcytic anemia and beta-thalassemia patients. The lowest blood catalase activities were detected in microcytic anemia and beta-thalassemia patients with the TT genotypes of the C111T polymorphism. This method was sensitive for detection of G113A acatalasemia mutation, but poorly detected C37T and G5A acatalasemia mutations. PMID:22286031

  12. A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector

    SciTech Connect

    Wang, Z.; Zhou, W.; Srivastava, T.; La Rosa, C.; Mandarino, A.; Forman, S.J.; Zaia, J.A.; Britt, W.J.; Diamond, D.J.

    2008-08-01

    A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models shows that it can stimulate primary immunity against all three antigens in both the CD4{sup +} and CD8{sup +} T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4{sup +} and CD8{sup +} T cell subsets.

  13. Shank3-mutant mice lacking exon 9 show altered excitation/inhibition balance, enhanced rearing, and spatial memory deficit.

    PubMed

    Lee, Jiseok; Chung, Changuk; Ha, Seungmin; Lee, Dongmin; Kim, Do-Young; Kim, Hyun; Kim, Eunjoon

    2015-01-01

    Shank3 is a postsynaptic scaffolding protein implicated in synapse development and autism spectrum disorders. The Shank3 gene is known to produce diverse splice variants whose functions have not been fully explored. In the present study, we generated mice lacking Shank3 exon 9 (Shank3 (Δ9) mice), and thus missing five out of 10 known Shank3 splice variants containing the N-terminal ankyrin repeat region, including the longest splice variant, Shank3a. Our X-gal staining results revealed that Shank3 proteins encoded by exon 9-containing splice variants are abundant in upper cortical layers, striatum, hippocampus, and thalamus, but not in the olfactory bulb or cerebellum, despite the significant Shank3 mRNA levels in these regions. The hippocampal CA1 region of Shank3 (Δ9) mice exhibited reduced excitatory transmission at Schaffer collateral synapses and increased frequency of spontaneous inhibitory synaptic events in pyramidal neurons. In contrast, prelimbic layer 2/3 pyramidal neurons in the medial prefrontal cortex displayed decreased frequency of spontaneous inhibitory synaptic events, indicating alterations in the ratio of excitation/inhibition (E/I ratio) in the Shank3 (Δ9) brain. These mice displayed a mild increase in rearing in a novel environment and mildly impaired spatial memory, but showed normal social interaction and repetitive behavior. These results suggest that ankyrin repeat-containing Shank3 splice variants are important for E/I balance, rearing behavior, and spatial memory.

  14. An Enhancer Near ISL1 and an Ultraconserved Exon of PCBP2 areDerived from a Retroposon

    SciTech Connect

    Bejerano, Gill; Lowe, Craig; Ahituv, Nadav; King, Bryan; Siepel,Adam; Salama, Sofie; Rubin, Edward M.; Kent, W. James; Haussler, David

    2005-11-27

    Hundreds of highly conserved distal cis-regulatory elementshave been characterized to date in vertebrate genomes1. Many thousandsmore are predicted based on comparative genomics2,3. Yet, in starkcontrast to the genes they regulate, virtually none of these regions canbe traced using sequence similarity in invertebrates, leaving theirevolutionary origin obscure. Here we show that a class of conserved,primarily non-coding regions in tetrapods originated from a novel shortinterspersed repetitive element (SINE) retroposon family that was activein Sarcopterygii (lobe-finned fishes and terrestrial vertebrates) in theSilurian at least 410 Mya4, and, remarkably, appears to be recentlyactive in the "living fossil" Indonesian coelacanth, Latimeriamenadoensis. We show that one copy is a distal enhancer, located 500kbfrom the neuro-developmental gene ISL1. Several others represent new,possibly regulatory, alternatively spliced exons in the middle ofpre-existing Sarcopterygian genes. One of these is the>200bpultraconserved region5, 100 percent identical in mammals, and 80 percentidentical to the coelacanth SINE, that contains a 31aa alternativelyspliced exon of the mRNA processing gene PCBP26. These add to a growinglist of examples7 in which relics of transposable elements have acquireda function that serves their host, a process termed "exaptation"8, andprovide an origin for at least some of the highly-conservedvertebrate-specific genomic sequences recently discovered usingcomparative genomics.

  15. From Blackboard to Green Screen: Teachers, Technology and Turmoil. Proceedings of the Conference of the Townsville Regional Group of the Australian College of Education (8th, Townsville, Queensland, Australia, May 15-16, 1987).

    ERIC Educational Resources Information Center

    Moon, R., Ed.

    The examination of technological change and its impact on schools that is reported in these conference proceedings focuses on the human dimensions of this change, and in particular, whether the introduction of technology into education is causing turmoil for teachers. It is argued that teachers appear to be feeling the strain of the actual changes…

  16. The exon quantification pipeline (EQP): a comprehensive approach to the quantification of gene, exon and junction expression from RNA-seq data

    PubMed Central

    Schuierer, Sven; Roma, Guglielmo

    2016-01-01

    The quantification of transcriptomic features is the basis of the analysis of RNA-seq data. We present an integrated alignment workflow and a simple counting-based approach to derive estimates for gene, exon and exon–exon junction expression. In contrast to previous counting-based approaches, EQP takes into account only reads whose alignment pattern agrees with the splicing pattern of the features of interest. This leads to improved gene expression estimates as well as to the generation of exon counts that allow disambiguating reads between overlapping exons. Unlike other methods that quantify skipped introns, EQP offers a novel way to compute junction counts based on the agreement of the read alignments with the exons on both sides of the junction, thus providing a uniformly derived set of counts. We evaluated the performance of EQP on both simulated and real Illumina RNA-seq data and compared it with other quantification tools. Our results suggest that EQP provides superior gene expression estimates and we illustrate the advantages of EQP's exon and junction counts. The provision of uniformly derived high-quality counts makes EQP an ideal quantification tool for differential expression and differential splicing studies. EQP is freely available for download at https://github.com/Novartis/EQP-cluster. PMID:27302131

  17. Assessing The Evolutionary Diversity Of Exon Skipping Events In Human, Mouse And Rat

    NASA Astrophysics Data System (ADS)

    Hsu, Fang-Rong; Chen, Chao-Jung; Kuo, Min-Chieh; Chang, Hwan-You; Shia, Wei-Chung

    2008-01-01

    This study is to research on the cross-species comparative analysis of homologous genetic sequence among human, mouse and rat by bioinformatics method, hopefully assessing the evolutionary diversity through exon length, reading frame preservation and KA/KS ratio test of alternative splicing events. Alternative splicing (AS) is an important mechanism in eukaryotic organism. We choose the "exon skipping events" from AS events for research. In the data of "conserved exon skipping events", we get 668 human-mouse conserved events, 179 human-rat conserved events and 266 conserved mouse-rat events. There are some extra data such as "non-conserved exon skipping events" and "species-specific events". We found out that the length of AS exon is shorter in conserved exon skipping event, but the ratio of reading frame preservation is higher. Among them, the minor form is the most special. We even got the same result in non-conserved exon skipping events. We calculated the KA/KS value by KA/KS ratio test and found out that the human-mouse KA/KS ratio is 0.158, the human-rat is 0.182 and the mouse-rat is 0.190. This represents that the human-mouse conserved events have the highest purifying selection pressure. In the end, we adopt KA/KS ratio test to do a further analysis between conserved and non-conserved exon skipping events and evaluate the evolutionary diversity of cross-species comparation.

  18. Lessons learnt from large-scale exon re-sequencing of the X chromosome

    PubMed Central

    Raymond, F. Lucy; Whibley, Annabel; Stratton, Michael R.; Gecz, Jozef

    2009-01-01

    A candidate gene approach to identifying novel causes of disease is concept-limiting and in the new era of high throughput sequencing there is now no need to restrict the experiment to a few interesting genes. We have recently completed a large-scale exon re-sequencing project using Sanger sequencing technology to analyse approximately 1 Mb of coding sequence of the X chromosome in probands from >200 families with various forms of intellectual disability. We review the lessons learnt from this experience. Comparing large data sets will certainly reveal pathogenic mutations in genes that were not possible to identify previously. However, the task of distinguishing pathogenic mutations from rare sequence variants is not easy and is the most substantial challenge to the next decade. High-throughput technology has the attraction of being cheap, fast and comprehensive but for projects that require detailed coverage of a genomic region at an exhaustive level they may require a combination of large-scale with a small-scale follow-up of difficult regions to sequence. The number of rare truncating variants present in coding regions of the X chromosome that are not pathogenic was 1%. The importance of the quality of the starting material both clinically and molecularly and the number of sequence variants both rare and common that any one individual has across their coding sequence is discussed. PMID:19297402

  19. Characterization of the exon structure of the Menkes disease gene using vectorette PCR

    SciTech Connect

    Tuemer, Z.; Tonnesen, T.; Horn, N.

    1995-04-10

    The gene defective in Menkes disease, an X-linked recessive disturbance of copper metabolism, has been isolated and predicted to encode a copper-binding P-type ATPase. We determined the complete exon-intron structure of the Menkes disease gene, which spans about 150 kb of genomic DNA. The gene contains 23 exons, and the ATG start codon is in the second exon. All of the exon-intron boundaries were sequenced and conformed to the GT/AT rule, except for the 5{prime} splice site of intron 9. A preliminary comparison demonstrated a striking similarity between the exon structures of the Menkes and Wilson disease genes, giving insight into their evolution. 33 refs., 3 figs., 2 tabs.

  20. The exon-intron organization of the human erythroid [beta]-spectrin gene

    SciTech Connect

    Amin, K.M.; Forget, B.G. ); Scarpa, A.L.; Curtis, P.J. ); Winkelmann, J.C. )

    1993-10-01

    The human erythrocyte [beta]-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid [beta]-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the [beta]-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present [beta]-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat [beta]10 is encoded by 4 exons. No single position of an intron in the [beta]-spectrin gene is conserved between any of the 17 [beta]-spectrin and 22 [alpha]-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5[prime] donor splice-site sequence begins with GC. The [beta]-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid [beta]-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp. 55 refs., 3 figs., 3 tabs.

  1. Microsomal epoxide hydrolase (EPHX1), slow (exon 3, 113His) and fast (exon 4, 139Arg) alleles confer susceptibility to squamous cell esophageal cancer

    SciTech Connect

    Jain, Meenu; Tilak, Anup Raj; Upadhyay, Rohit; Kumar, Ashwani; Mittal, Balraj

    2008-07-15

    Genetic polymorphisms in xenobiotic metabolizing enzymes may alter risk of various cancers. Present case-control study evaluated the influence of EPHX1 genetic variations on squamous cell esophageal cancer (ESCC) susceptibility in 107 patients and 320 controls. EPHX1 polymorphic alleles were genotyped by direct sequencing (exon 3, Tyr113His) or PCR-RFLP (exon 4, His139Arg). Patients with exon 3 genotypes (Tyr113His, His113His) and 113His allele were at risk of ESCC (OR{sub Tyr113His} 2.0, 95% CI = 1.2-3.4, p = 0.007; OR{sub His113His} 2.3 95% CI = 1.0-5.2, p = 0.03 and OR{sub His} 1.5, 95% CI = 1.0-2.1, p = 0.01). In contrast, individuals with exon 4, 139Arg allele were at low risk of cancer (OR 0.34, 95% CI = 0.20-0.56, p = 0.001). However, none of haplotype combinations of exon 3 (Tyr113His) and exon 4 (His139Arg) polymorphisms showed modulation of risk for ESCC. Sub-grouping of patients based on anatomical location of tumor predicted that patients with exon 3, His113His and Tyr113His genotypes were at higher risk for developing ESCC tumor at upper and middle third locations (OR 4.4, 95% CI = 1.0-18.5, p = 0.04; OR 2.5, 95% CI = 1.3-5.0, p = 0.005 respectively). The frequency of exon 4, His139Arg genotype was significantly lower in ESCC patients with lower third tumor location as compared to controls (14.8% vs. 36.3%, p = 0.02). In case-only study, gene-environment interaction of EPHX1 genotypes with tobacco, alcohol and occupational exposures did not appear to modulate the cancer susceptibility. In conclusion, exon 3, Tyr113His genotype was associated with higher risk of ESCC particularly at upper and middle-third anatomical locations of tumor. However, His139Arg genotype of exon 4, exhibited low risk for ESCC as well as its clinical characteristics.

  2. Differential methylation of the promoter and first exon of the RASSF1A gene in hepatocarcinogenesis

    PubMed Central

    Jain, Surbhi; Xie, Lijia; Boldbaatar, Batbold; Lin, Selena Y.; Hamilton, James P.; Meltzer, Stephen J.; Chen, Shun-Hua; Hu, Chi-Tan; Block, Timothy M.; Song, Wei; Su, Ying-Hsiu

    2015-01-01

    Aim Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening. Methods Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening. Results In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC. Conclusion Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC. PMID:25382672

  3. TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping.

    PubMed

    Maydan, G; Andresen, B S; Madsen, P P; Zeigler, M; Raas-Rothschild, A; Zlotogorski, A; Gutman, A; Korman, S H

    2006-10-01

    Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner-Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon-intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5' donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue.

  4. Becker Muscular Dystrophy (BMD) caused by duplication of exons 3-6 of the dystrophin gene presenting as dilated cardiomyopathy

    SciTech Connect

    Tsai, A.C.; Allingham-Hawkins, D.J.; Becker, L.

    1994-09-01

    X-linked dilated cardiomyopathy (XLCM) is a progressive myocardial disease presenting with congestive heart failure in teenage males without clinical signs of skeletal myopathy. Tight linkage of XLCM to the DMD locus has been demonstrated; it has been suggested that, at least in some families, XLCM is a {open_quotes}dystrophinopathy.{close_quotes} We report a 14-year-old boy who presented with acute heart failure due to dilated cardiomyopathy. He had no history of muscle weakness, but physical examination revealed pseudohypertrophy of the calf muscles. He subsequently received a heart transplantation. Family history was negative. Serum CK level at the time of diagnosis was 10,416. Myocardial biopsy showed no evidence of carditis. Dystrophin staining of cardiac and skeletal muscle with anti-sera to COOH and NH{sub 2}termini showed a patchy distribution of positivity suggestive of Becker muscular dystrophy. Analysis of 18 of the 79 dystrophin exons detected a duplication that included exons 3-6. The proband`s mother has an elevated serum CK and was confirmed to be a carrier of the same duplication. A mutation in the muscle promotor region of the dystrophin gene has been implicated in the etiology of SLCM. However, Towbin et al. (1991) argued that other 5{prime} mutations in the dystrophin gene could cause selective cardiomyopathy. The findings in our patient support the latter hypothesis. This suggests that there are multiple regions in the dystrophin gene which, when disrupted, can cause isolated dilated cardiomyopathy.

  5. The exon-junction-complex-component metastatic lymph node 51 functions in stress-granule assembly.

    PubMed

    Baguet, Aurélie; Degot, Sébastien; Cougot, Nicolas; Bertrand, Edouard; Chenard, Marie-Pierre; Wendling, Corinne; Kessler, Pascal; Le Hir, Hervé; Rio, Marie-Christine; Tomasetto, Catherine

    2007-08-15

    Metastatic lymph node 51 [MLN51 (also known as CASC3)] is a component of the exon junction complex (EJC), which is assembled on spliced mRNAs and plays important roles in post-splicing events. The four proteins of the EJC core, MLN51, MAGOH, Y14 and EIF4AIII shuttle between the cytoplasm and the nucleus. However, unlike the last three, MLN51 is mainly detected in the cytoplasm, suggesting that it plays an additional function in this compartment. In the present study, we show that MLN51 is recruited into cytoplasmic aggregates known as stress granules (SGs) together with the SG-resident proteins, fragile X mental retardation protein (FMRP), poly(A) binding protein (PABP) and poly(A)(+) RNA. MLN51 specifically associates with SGs via its C-terminal region, which is dispensable for its incorporation in the EJC. MLN51 does not promote SG formation but its silencing, or the overexpression of a mutant lacking its C-terminal region, alters SG assembly. Finally, in human breast carcinomas, MLN51 is sometimes present in cytoplasmic foci also positive for FMRP and PABP, suggesting that SGs formation occurs in malignant tumours.

  6. Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping.

    PubMed

    Gao, Quan Q; Wyatt, Eugene; Goldstein, Jeff A; LoPresti, Peter; Castillo, Lisa M; Gazda, Alec; Petrossian, Natalie; Earley, Judy U; Hadhazy, Michele; Barefield, David Y; Demonbreun, Alexis R; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M

    2015-11-01

    Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.

  7. The first exon duplication mouse model of Duchenne muscular dystrophy: A tool for therapeutic development.

    PubMed

    Vulin, Adeline; Wein, Nicolas; Simmons, Tabatha R; Rutherford, Andrea M; Findlay, Andrew R; Yurkoski, Jacqueline A; Kaminoh, Yuuki; Flanigan, Kevin M

    2015-11-01

    Exon duplication mutations account for up to 11% of all cases of Duchenne muscular dystrophy (DMD), and a duplication of exon 2 is the most common duplication in patients. For use as a platform for testing of duplication-specific therapies, we developed a mouse model that carries a Dmd exon 2 duplication. By using homologous recombination we duplicated exon 2 within intron 2 at a location consistent with a human duplication hotspot. mRNA analysis confirms the inclusion of a duplicated exon 2 in mouse muscle. Dystrophin expression is essentially absent by immunofluorescent and immunoblot analysis, although some muscle specimens show very low-level trace dystrophin expression. Phenotypically, the mouse shows similarities to mdx, the standard laboratory model of DMD. In skeletal muscle, areas of necrosis and phagocytosis are seen at 3 weeks, with central nucleation prominent by four weeks, recapitulating the "crisis" period in mdx. Marked diaphragm fibrosis is noted by 6 months, and remains unchanged at 12 months. Our results show that the Dup2 mouse is both pathologically (in degree and distribution) and physiologically similar to mdx. As it recapitulates the most common single exon duplication found in DMD patients, this new model will be a useful tool to assess the potential of duplicated exon skipping.

  8. The Evolutionary Fate of Alternatively Spliced Homologous Exons after Gene Duplication

    PubMed Central

    Abascal, Federico; Tress, Michael L.; Valencia, Alfonso

    2015-01-01

    Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene. PMID:25931610

  9. Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping.

    PubMed

    Gao, Quan Q; Wyatt, Eugene; Goldstein, Jeff A; LoPresti, Peter; Castillo, Lisa M; Gazda, Alec; Petrossian, Natalie; Earley, Judy U; Hadhazy, Michele; Barefield, David Y; Demonbreun, Alexis R; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M

    2015-11-01

    Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations. PMID:26457733

  10. Comparison of exon 5 sequences from 35 class I genes of the BALB/c mouse

    PubMed Central

    1989-01-01

    DNA sequences of the fifth exon, which encodes the transmembrane domain, were determined for the BALB/c mouse class I MHC genes and used to study the relationships between them. Based on nucleotide sequence similarity, the exon 5 sequences can be divided into seven groups. Although most members within each group are at least 80% similar to each other, comparison between groups reveals that the groups share little similarity. However, in spite of the extensive variation of the fifth exon sequences, analysis of their predicted amino acid translations reveals that only four class I gene fifth exons have frameshifts or stop codons that terminate their translation and prevent them from encoding a domain that is both hydrophobic and long enough to span a lipid bilayer. Exactly 27 of the remaining fifth exons could encode a domain that is similar to those of the transplantation antigens in that it consists of a proline-rich connecting peptide, a transmembrane segment, and a cytoplasmic portion with membrane- anchoring basic residues. The conservation of this motif in the majority of the fifth exon translations in spite of extensive variation suggests that selective pressure exists for these exons to maintain their ability to encode a functional transmembrane domain, raising the possibility that many of the nonclassical class I genes encode functionally important products. PMID:2584927

  11. Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Ma, Ruiqin; He, Feng; Wen, Haishen; Li, Jifang; Shi, Bao; Shi, Dan; Liu, Miao; Mu, Weijie; Zhang, Yuanqing; Hu, Jian; Han, Weiguo; Zhang, Jianan; Wang, Qingqing; Yuan, Yuren; Liu, Qun

    2012-03-01

    As a specific gene of fish, cytochrome P450c17-II ( CYP17-II) gene plays a key role in the growth, development an reproduction level of fish. In this study, the single-stranded conformational polymorphism (SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-II gene in a population of 75 male Japanese flounder ( Paralichthys olivaceus). Three single nucleotide polymorphisms (SNPs) were identified in CYP17-II gene of Japanese flounder. They were c.G594A (p.G188R), c.G939A and c.G1502A (p.G490D). SNP1 (c.G594A), located in exon 4 of CYP17-II gene, was significantly associated with gonadosomatic index (GSI). Individuals with genotype GG of SNP1 had significantly lower GSI ( P < 0.05) than those with genotype AA or AG. SNP2 (c.G939A) located at the CpG island of CYP17-II gene. The mutation changed the methylation of exon 6. Individuals with genotype AA of SNP2 had significantly lower serum testosterone (T) level and hepatosomatic index (HSI) compared to those with genotype GG. The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder. However, the SNP3 (c.G1502A) located in exon 9 did not affect the four measured reproductive traits. This study showed that CYP17-II gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

  12. A Cross-Platform Comparison of Genome-Wide Expression Changes of Laser Microdissected Lung Tissue of C-Raf Transgenic Mice Using 3′IVT and Exon Array

    PubMed Central

    Londhe, Kishor Bapu; Borlak, Juergen

    2012-01-01

    Microarrays are widely used to study genome-wide gene expression changes in different conditions most notably disease, growth, or to investigate the effects of drugs on entire genomes. While the number and gene probe sequences to investigate individual gene expression changes differs amongst manufactures, the design for all of the probes is biased towards the 3′ region. With the advent of exon arrays, transcripts of any known or predicted exon can be investigated to facilitate the study of genome-wide alternative splicing events. Thus, the use of exon arrays provides unprecedented opportunities in gene expression studies. However, it remains a major challenge to directly compare gene expression data derived from oligonucleotide to exon arrays. In the present study, genome-wide expression profiling of Laser Micro-dissected Pressure Catapulted (LMPC) samples of c-Raf mouse lung adenocarcinoma, dysplasia, unaltered transgenic and non-transgenic tissues was performed using the Affymetrix GeneChip Mouse Genome 430 2.0 Array and whole genome Mouse Exon 1.0 ST Array. Based on individual group comparisons 52 to 83% of regulated genes were similar in direction, but fold changes of regulated genes disagreed when data amongst the two platforms were compared. Furthermore, for 27 regulated genes opposite direction of gene expression was observed when the two platforms were compared pointing to the need to assess alternative splicing events at the 3′ end. Taken collectively, exon arrays can be performed even with laser microdissected samples but fold change gene expression changes differ considerably between 3′IVT array and exon arrays with alternative splicing events contributing to apparent differences in gene expression changes. PMID:22815814

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

    PubMed Central

    Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

    2012-01-01

    Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3′ and 5′ splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

  14. The expression of the human steroid sulfatase-encoding gene is driven by alternative first exons.

    PubMed

    Dalla Valle, Luisa; Toffolo, Vania; Nardi, Alessia; Fiore, Cristina; Armanini, Decio; Belvedere, Paola; Colombo, Lorenzo

    2007-10-01

    We have analyzed steroid sulfatase (STS) gene transcription in 10 human tissues: ovary, adrenal cortex, uterus, thyroid, liver, pancreas, colon, mammary gland, dermal papilla of the hair follicle, and peripheral mononuclear leukocytes. Overall, six different promoters were found to drive STS expression, giving rise to transcripts with unique first exons that were labeled 0a, 0b, 0c, 1a, 1c, and 1d, of which the last two and 0c are newly reported. All of them, except exon 1d, vary in length owing to the occurrence of multiple transcriptional start sites. While placental exon 1a is partially coding, the other five first exons are all untranslated. Three of these (0a, 0b, and 0c) are spliced to the common partially coding exon 1b, whereas the other two (1c and 1d) are spliced to the coding exon 2, which occurs in all transcripts. Whatever the ATG actually used, the differences are restricted to the signal peptide which is post-transcriptionally cleaved. Transcripts with exons 0a and 0b have the broadest tissue distribution, occurring, in 6 out of the 12 tissues so far investigated, while the other first exons are restricted to one or two tissues. The proximal promoter of each first exon was devoid of TATA box or initiator element and lacked consensus elements for transcription factors related to steroidogenesis, suggesting that regulatory sequences are probably placed at greater distance. In conclusion, the regulation of STS transcription appears to be more complex than previously thought, suggesting that this enzyme plays a substantial role in intercellular integration. PMID:17601726

  15. Increased damage of exon 5 of p53 gene in workers from an arsenic plant.

    PubMed

    Wen, Weihua; Che, Wangjun; Lu, Lin; Yang, Jun; Gao, Xufang; Wen, Jinghua; Heng, Zhengchang; Cao, Shuqiao; Cheng, Huirong

    2008-08-25

    Mutagenesis is a multistage process. Substitution mutations can be induced by base modified through alteration of pairing property. Mutations of exon 5 and 8 of p53 gene have been found in most arsenicosis patients with precarcinomas and carcinomas, but never in arsenicosis individuals without precarcinomas and carcinomas. This study investigates whether base modification exists in exon 5 and 8 of p53 gene, and explores the dose-effect relationship between damage of exon 5 of p53 gene and urinary arsenic. Concentrations of urinary 8-hydroxydeoxyguanine (8-OHdG) are analyzed to identify the occurrence of DNA damage. The real-time PCR developed by Sikorsky et al. is applied to detect base modification in exon 5 and 8 of p53 gene for apparently healthy participants. Our results show that the mean total arsenic concentrations of two exposed groups from an arsenic plant are significantly elevated compared with the control group, and the damage level of exon 5 of the high-exposed group is significantly higher than that of the control group, but which does not happen in exon 8. The closely correlation between the damage index of exon 5 and urinary organic arsenic concentration are found. Concentration of 8-OHdG of the high-exposed group is significantly higher than that of the control group. These results imply that base modification in exon 5 of p53 gene can be induced by arsenic. In addition, our study suggests that the damage level of exon 5 is a useful biomarker to assess adverse health effect levels caused by chronic exposure to arsenic. PMID:18621066

  16. Increased damage of exon 5 of p53 gene in workers from an arsenic plant.

    PubMed

    Wen, Weihua; Che, Wangjun; Lu, Lin; Yang, Jun; Gao, Xufang; Wen, Jinghua; Heng, Zhengchang; Cao, Shuqiao; Cheng, Huirong

    2008-08-25

    Mutagenesis is a multistage process. Substitution mutations can be induced by base modified through alteration of pairing property. Mutations of exon 5 and 8 of p53 gene have been found in most arsenicosis patients with precarcinomas and carcinomas, but never in arsenicosis individuals without precarcinomas and carcinomas. This study investigates whether base modification exists in exon 5 and 8 of p53 gene, and explores the dose-effect relationship between damage of exon 5 of p53 gene and urinary arsenic. Concentrations of urinary 8-hydroxydeoxyguanine (8-OHdG) are analyzed to identify the occurrence of DNA damage. The real-time PCR developed by Sikorsky et al. is applied to detect base modification in exon 5 and 8 of p53 gene for apparently healthy participants. Our results show that the mean total arsenic concentrations of two exposed groups from an arsenic plant are significantly elevated compared with the control group, and the damage level of exon 5 of the high-exposed group is significantly higher than that of the control group, but which does not happen in exon 8. The closely correlation between the damage index of exon 5 and urinary organic arsenic concentration are found. Concentration of 8-OHdG of the high-exposed group is significantly higher than that of the control group. These results imply that base modification in exon 5 of p53 gene can be induced by arsenic. In addition, our study suggests that the damage level of exon 5 is a useful biomarker to assess adverse health effect levels caused by chronic exposure to arsenic.

  17. Exon skipping through the creation of a putative exonic splicing silencer as a consequence of the cystic fibrosis mutation R553X.

    PubMed

    Aznarez, Isabel; Zielenski, Julian; Rommens, Johanna M; Blencowe, Benjamin J; Tsui, Lap-Chee

    2007-05-01

    Nonsense mutations that occur more than 50 bases upstream of terminal spliced junctions are generally thought to lead to degradation of the corresponding transcripts by the process of nonsense-mediated mRNA decay. It has also been proposed that some nonsense mutations may affect splicing by the process of nonsense-associated altered splicing (NAS), or by the disruption of a splicing regulatory element. In this study, the effect of the R553X mutation on the splicing of exon 11 of the cystic fibrosis transmembrane conductance regulator gene was investigated. Evidence that R553X causes exon 11 to skip through the creation of a putative exonic splicing silencer (ESS) was provided. The putative ESS appears to be active when located immediately upstream of a 5' splice site. These findings argue against the possibility that R553X-associated exon 11 skipping is caused by NAS. The study further suggests that aminoglycoside antibiotic treatment would not be effective for patients with the R553X mutation, owing to the skipping of exon 11, and further emphasises the need for detailed mechanistic characterisation of the consequences of nonsense disease mutations. PMID:17475917

  18. Intron-exon organization of the active human protein S gene PS. alpha. and its pseudogene PS. beta. : Duplication and silencing during primate evolution

    SciTech Connect

    Ploos van Amstel, H.; Reitsma, P.H.; van der Logt, C.P.; Bertina, R.M. )

    1990-08-28

    The human protein S locus on chromosome 3 consists of two protein S genes, PS{alpha} and PS{beta}. Here the authors report the cloning and characterization of both genes. Fifteen exons of the PS{alpha} gene were identified that together code for protein S mRNA as derived from the reported protein S cDNAs. Analysis by primer extension of liver protein S mRNA, however, reveals the presence of two mRNA forms that differ in the length of their 5{prime}-noncoding region. Both transcripts contain a 5{prime}-noncoding region longer than found in the protein S cDNAs. The two products may arise from alternative splicing of an additional intron in this region or from the usage of two start sites for transcription. The intron-exon organization of the PS{alpha} gene fully supports the hypothesis that the protein S gene is the product of an evolutional assembling process in which gene modules coding for structural/functional protein units also found in other coagulation proteins have been put upstream of the ancestral gene of a steroid hormone binding protein. The PS{beta} gene is identified as a pseudogene. It contains a large variety of detrimental aberrations, viz., the absence of exon I, a splice site mutation, three stop codons, and a frame shift mutation. Overall the two genes PS{alpha} and PS{beta} show between their exonic sequences 96.5% homology. Southern analysis of primate DNA showed that the duplication of the ancestral protein S gene has occurred after the branching of the orangutan from the African apes. A nonsense mutation that is present in the pseudogene of man also could be identified in one of the two protein S genes of both chimpanzee and gorilla. This implicates that silencing of one of the two protein S genes must have taken place before the divergence of the three African apes.

  19. Genetic associations of nonsynonymous exonic variants with psychophysiological endophenotypes.

    PubMed

    Vrieze, Scott I; Malone, Stephen M; Pankratz, Nathan; Vaidyanathan, Uma; Miller, Michael B; Kang, Hyun Min; McGue, Matt; Abecasis, Gonçalo; Iacono, William G

    2014-12-01

    We mapped ∼85,000 rare nonsynonymous exonic single nucleotide polymorphisms (SNPs) to 17 psychophysiological endophenotypes in 4,905 individuals, including antisaccade eye movements, resting EEG, P300 amplitude, electrodermal activity, affect-modulated startle eye blink. Nonsynonymous SNPs are predicted to directly change or disrupt proteins encoded by genes and are expected to have significant biological consequences. Most such variants are rare, and new technologies can efficiently assay them on a large scale. We assayed 247,870 mostly rare SNPs on an Illumina exome array. Approximately 85,000 of the SNPs were polymorphic, rare (MAF < .05), and nonsynonymous. Single variant association tests identified a SNP in the PARD3 gene associated with theta resting EEG power. The sequence kernel association test, a gene-based test, identified a gene PNPLA7 associated with pleasant difference startle, the difference in startle magnitude between pleasant and neutral images. No other single nonsynonymous variant, or gene-based group of variants, was strongly associated with any endophenotype. PMID:25387709

  20. Genetic associations of nonsynonymous exonic variants with psychophysiological endophenotypes

    PubMed Central

    Vrieze, Scott I.; Malone, Stephen M.; Pankratz, Nathan; Vaidyanathan, Uma; Miller, Michael B.; Kang, Hyun Min; McGue, Matt; Abecasis, Gonçalo; Iacono, William G.

    2014-01-01

    We mapped ~85,000 rare nonsynonymous exonic single nucleotide polymorphisms (SNPs) to 17 psychophysiological endophenotypes in 4,905 individuals, including antisaccade eye movements, resting EEG, P300 amplitude, electrodermal activity, affect-modulated startle eye blink. Nonsynonymous SNPs are predicted to directly change or disrupt proteins encoded by genes and are expected to have significant biological consequences. Most such variants are rare, and new technologies can efficiently assay them on a large scale. We assayed 247,870 mostly rare SNPs on an Illumina exome array. Approximately 85,000 of the SNPs were polymorphic, rare (MAF < .05), and nonsynonymous. Single variant association tests identified a SNP in the PARD3 gene associated with theta resting EEG power. The sequence kernel association test, a gene-based test, identified a gene PNPLA7 associated with pleasant difference startle, the difference in startle magnitude between pleasant and neutral images. No other single nonsynonymous variant, or gene-based group of variants, was strongly associated with any endophenotype. PMID:25387709

  1. Dipole entropy based techniques for segmentation of introns and exons in DNA

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, Nithya; Bose, R.

    2012-08-01

    We have used superinformation, which is a measure of the disorder of the entropy content of different portions of a sequence, to analyze the structural variations of the introns and exons in DNA. We have computed superinformation for the angles of the dipole moments of the base-pairs and nucleotides in the double and single-stranded forms of DNA, respectively. We show that the computed dipole-angular superinformation of the introns are significantly higher than those of the exons and that these techniques could be used for intron-exon segmentation. They also yield more accurate and computationally faster results than the previously reported methods.

  2. Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network

    PubMed Central

    Beau-Faller, M.; Prim, N.; Ruppert, A.-M.; Nanni-Metéllus, I.; Lacave, R.; Lacroix, L.; Escande, F.; Lizard, S.; Pretet, J.-L.; Rouquette, I.; de Crémoux, P.; Solassol, J.; de Fraipont, F.; Bièche, I.; Cayre, A.; Favre-Guillevin, E.; Tomasini, P.; Wislez, M.; Besse, B.; Legrain, M.; Voegeli, A.-C.; Baudrin, L.; Morin, F.; Zalcman, G.; Quoix, E.; Blons, H.; Cadranel, J.

    2014-01-01

    Background There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. Patients and methods EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. Results Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12–24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08–0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6–21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). Conclusions Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment. PMID:24285021

  3. Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene.

    PubMed Central

    Mäkelä, T P; Saksela, K; Alitalo, K

    1989-01-01

    The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance

  4. Deletion mutations of the Bs-alpha gene in patients with Albright hereditary osteodystrophy: Possible mutation hot-spot in exon 7

    SciTech Connect

    Weinstein, L.S.; Hainline, B.E.; Schuster, V.

    1994-09-01

    Albright hereditary osteodystrophy (AHO) is an autosomal dominant disease characterized by short stature, centripetal obesity, subcutaneous ossifications and focal brachydactyly. Patients with this disorder may have these features alone (pseudopseudohypoparathyroidism) or these features in association with resistance to multiple hormones which raise intracellular cAMP (pseudohypoparathyroidism, PHP). In most kindreds, affected members have decreased function of the G protein Gs and decreased levels of the Gs{alpha}-subunit. Heterozygous inactivating mutations of the Gs{alpha} gene have been previously identified in AHO. Exons 2-13 of the Gs{alpha} gene and their splice junctions were PCR-amplified and the products analyzed by temperature gradient gel electrophoresis (TGGE) and direct sequencing. Using this approach, a new heterozygous 2 bp deletion in exon 4 creating a premature stop codon was identified in 5 affected members of a previously reported family. The mutation was not present in an unaffected family member. We also have identified a previously reported 4 bp deletion in the coding region of exon 7 in 2 further unrelated sporadic cases of PHP. In one case, the mutation was absent in her siblings and in both parents, confirming that this is a de novo mutation in this patient. This specific 4 bp deletion has now been reported in 4 PHP patients, at least 3 of whom are unrelated. These results suggest that this region of the Gs{alpha} gene may be a hot-spot for deletions.

  5. One Novel Frameshift Mutation on Exon 64 of COL7A1 Gene in an Iranian Individual Suffering Recessive Dystrophic Epidermolysis Bullosa.

    PubMed

    Khaniani, Mahmoud Shekari; Sohrabi, Nasrin; Derakhshan, Neda Mansoori; Derakhshan, Sima Mansoori

    2015-01-01

    Recessive dystrophic epidermolysis bullosa (RDEB) is an extremely rare subtype of bullous dermatosis caused by the COL7A1 gene mutation. After genomic DNA extraction from the peripheral blood sample of all subjects (3 pedigree members and 3 unrelated control individuals), COL7A1 gene screening was performed by PCR amplification and direct DNA sequencing of all of the coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in an affected individual revealed a novel mutation: c.5493delG (p.K1831Nfs*10) in exon 64 of the COL7A1 gene in homozygous state. This mutation was not discovered in 3 unrelated Iranian control individuals. These data suggest that c.5493delG may influence the phenotype of RDEB. The result of this case report contributes to the expanding database on COL7A1 mutations.

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

    PubMed

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

    2014-09-01

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

  7. Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene

    PubMed Central

    Flanigan, Kevin M.; Dunn, Diane M.; von Niederhausern, Andrew; Soltanzadeh, Payam; Howard, Michael T.; Sampson, Jacinda B.; Swoboda, Kathryn J.; Bromberg, Mark B.; Mendell, Jerry R.; Taylor, Laura; Anderson, Christine B.; Pestronk, Alan; Florence, Julaine; Connolly, Anne M.; Mathews, Katherine D.; Wong, Brenda; Finkel, Richard S.; Bonnemann, Carsten G.; Day, John W.; McDonald, Craig; Weiss, Robert B.

    2013-01-01

    Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping. PMID:21972111

  8. Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene.

    PubMed

    Flanigan, Kevin M; Dunn, Diane M; von Niederhausern, Andrew; Soltanzadeh, Payam; Howard, Michael T; Sampson, Jacinda B; Swoboda, Kathryn J; Bromberg, Mark B; Mendell, Jerry R; Taylor, Laura E; Anderson, Christine B; Pestronk, Alan; Florence, Julaine M; Connolly, Anne M; Mathews, Katherine D; Wong, Brenda; Finkel, Richard S; Bonnemann, Carsten G; Day, John W; McDonald, Craig; Weiss, Robert B

    2011-03-01

    Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping.

  9. Recurring exon deletions in the haptoglobin (HP) gene associate with lower blood cholesterol levels

    PubMed Central

    Boettger, Linda M.; Salem, Rany M.; Handsaker, Robert E.; Peloso, Gina; Kathiresan, Sekar; Hirschhorn, Joel; McCarroll, Steven A.

    2016-01-01

    Two exons of the human haptoglobin (HP) gene exhibit copy number variation that affects HP multimerization and underlies one of the first protein polymorphisms identified in humans. The evolutionary origins and medical significance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from the recurring reversion of an ancient hominin-specific duplication of these exons. Though this polymorphism has been largely invisible to genome-wide genetic studies to date, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We show that these deletions, and a SNP that affects HP expression, are the likely drivers of the strong but complex association of cholesterol levels to SNPs near HP. Recurring exonic deletions in the haptoglobin gene likely enhance human health by lowering cholesterol levels in the blood. PMID:26901066

  10. Identification of a tyrosinase (TYR) exon 4 deletion in albino ferrets (Mustela putorius furo).

    PubMed

    Blaszczyk, W M; Distler, C; Dekomien, G; Arning, L; Hoffmann, K-P; Epplen, J T

    2007-08-01

    Albinism is due to a lack of pigmentation in hair, skin and eye, and has been shown to occur in several animal species. Mutations of the tyrosinase (TYR) gene account for albinism in domestic cats, rabbits, cattle, mice and rats. In this study, we demonstrate that a TYR mutation accounts for albinism in the ferret (Mustela putorius furo). The coding sequence of the five exons of TYR was determined in genomic DNA from wild-type pigmented 'sable' coloured and albino ferrets. It was not possible to amplify TYR exon 4 in albino ferrets originating from different breeds. The deletion of exon 4 in albino ferrets was confirmed by Southern blot hybridization of genomic DNA from albino and pigmented ferrets. This is the first report of a deletion of a TYR exon in a non-human mammal. PMID:17655555

  11. Immunoglobulin VH genes are transcribed by T cells in association with a new 5' exon

    PubMed Central

    1988-01-01

    We previously detected mRNAs in a number of human T cell lines with a probe from within the Ig VH gene locus. We now show these mRNAs consist of Ig VH genes expressed in T cells. In one human T cell line, two RNA species have been studied and found to come from transcripts of unrearranged VH segments in which the leader exon, normally associated with VH transcripts in B cells, is replaced by a novel 5' exon (ET) not encoding a hydrophobic leader peptide. In genomic DNA, this new ET exon is adjacent to a pseudo-VH gene that has not been observed in mature mRNA. This implies that RNA splicing controls association of the new exon with the expressed VH segments. Hence, VH transcription does indeed occur in T cells, but is qualitatively different from that in B cells. PMID:3133445

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

    PubMed

    Trzcinska-Danielewicz, J; Fronk, J

    2000-09-15

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

  13. Identification of intron/exon boundaries in genomic DNA by inverse PCR.

    PubMed

    Albertsen, H; Thliveris, A

    2001-05-01

    This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template. This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. PMID:18428300

  14. Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.

    PubMed

    Furuyama, Kazuto; Harada, Taishi; Iwama, Eiji; Shiraishi, Yoshimasa; Okamura, Kyoko; Ijichi, Kayo; Fujii, Akiko; Ota, Keiichi; Wang, Shuo; Li, Heyan; Takayama, Koichi; Giaccone, Giuseppe; Nakanishi, Yoichi

    2013-05-01

    The presence of epidermal growth factor receptor (EGFR) somatic mutations in non-small-cell lung cancer patients is associated with response to treatment with EGFR-tyrosine kinase inhibitors, such as gefitinib and erlotinib. More than 100 mutations in the kinase domain of EGFR have been identified. In particular there are many variations of deletion mutations in exon 19. In this study, using yellow fluorescent protein-tagged fragments of the EGFR intracellular domain, we examined the differences in sensitivity to gefitinib, erlotinib and afatinib between several exon 19 mutants and other common EGFR mutations. We also used serum of patients undergoing treatment with EGFR-tyrosine kinase inhibitors in this system. In addition, we examined the relative kinase activity of these mutants by measuring relative fluorescent intensity after immunofluorescence staining. We found that both sensitivity to EGFR-tyrosine kinase inhibitors and relative kinase activity differed among several EGFR mutations found in the same region of the kinase domain. This study underscores the importance of reporting the clinical outcome of treatment in relation to different EGFR mutations.

  15. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    PubMed

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.

  16. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    PubMed

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process. PMID:26320575

  17. Computer analysis of protein functional sites projection on exon structure of genes in Metazoa

    PubMed Central

    2015-01-01

    Background Study of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem. Results One approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling. Conclusions These results characterize the features of the coding exon-intron structure that affect the

  18. Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene

    PubMed Central

    Seo, Joonbae; Singh, Natalia N.; Ottesen, Eric W.; Sivanesan, Senthilkumar; Shishimorova, Maria; Singh, Ravindra N.

    2016-01-01

    Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein. PMID:27111068

  19. Further genotype-phenotype correlation emerging from two families with PLP1 exon 4 skipping.

    PubMed

    Biancheri, Roberta; Grossi, Serena; Regis, Stefano; Rossi, Andrea; Corsolini, Fabio; Rossi, Daniela Paola; Cavalli, Pietro; Severino, Mariasavina; Filocamo, Mirella

    2014-03-01

    Proteolipid protein 1 (PLP1) gene-related disorders due to mutations in the PLP1 include a wide spectrum of X-linked disorders ranging from severe connatal Pelizaeus-Merzbacher disease (PMD) to spastic paraplegia 2 (SPG2). Duplications, deletions or point mutations in coding and noncoding regions of the PLP1 gene may occur. We report the clinical, neuroradiologic and molecular findings in six patients from two unrelated families. The affected males showed severe mental retardation, spastic tetraparesis, inability of walking and pes cavus at onset in early infancy. Brain magnetic resonance imaging (MRI) showed hypomyelination and brain atrophy. Nystagmus was never observed. The affected females showed adult-onset progressive spastic paraparesis leading to wheel-chair dependency and subtle white matter changes on brain MRI. Molecular studies in the two families identified two different intronic mutations, the novel c.622+2T>C and the known c.622+1G>A, leading to the skipping of PLP1-exon 4. The clinical presentation of the affected males did not consistently fit in any of the PLP1-related disorder subtypes (i.e., connatal or classic PMD, SPG2 and 'PLP1 null syndrome'), and in addition, the carrier females were symptomatic despite the severe clinical picture of their respective probands. This study provides new insight into the genotype-phenotype correlations of patients with PLP1 splice-site mutations. PMID:23711321

  20. POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation.

    PubMed

    Johansson, Anna-Mia; Stenberg, Per; Pettersson, Fredrik; Larsson, Jan

    2007-11-01

    Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional "peak" in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome.

  1. POF and HP1 Bind Expressed Exons, Suggesting a Balancing Mechanism for Gene Regulation

    PubMed Central

    Johansson, Anna-Mia; Stenberg, Per; Pettersson, Fredrik; Larsson, Jan

    2007-01-01

    Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional “peak” in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome. PMID:18020713

  2. Hereditary vitamin D resistant rickets due to deletion of exon 3 of the vitamin D receptor

    SciTech Connect

    Rut, A.R.; O`Riordan, J.L.H.; Hughes, M.R.

    1994-09-01

    Hereditary vitamin D resistant rickets is an autosomal recessive disorder characterized by severe rickets, hypolcalcaemia, secondary hyperparathyroidism and occasionally, the absence of body hair. The pathological process involves resistance of target tissues to the actions of calcitriol [1,25(OH{sub 2}D{sub 3})], the hormonal form of vitamin D. Calcitriol mediates its actions through a nuclear receptor (VDR) which has been cloned and shown to be a member of the superfamily of steriod/thyroid/retinoic acid receptors. Skin fibroblasts were obtained from a Greek child with characteristic features of the condition. Total RNA was extracted from rapidly dividing cells and reverse transcribed. The coding region was amplified by PCR with primers 31a in the 5{prime} untranslated region and 31b in the 3{prime} untranslated region of the VDR cDNA sequence. The 5{prime} and 3{prime} halves of VDR were further amplified using primers tagged with M13 forward and reverse primer sequences. The whole process was carried out in duplicate starting with RNA. Sequence data was obtained using Taq dye primer cycle sequencing (ABI). Agarose gel electrophoresis revealed that the 5{prime} product was approximately 100 bp shorter than control. This was confirmed by sequencing which demonstrated a 131 bp deletion of the C-terminal part of the DNA binding domain (bases 147-277). Bases 147-277 are coded for by exon 3 and this deletion is bounded by the splice junctions. This is the first report of a deletion in VDR in any patient with vitamin D-resistant rickets. Such a deletion not only removes the second zinc finger but also results in a frameshift that corrupts the remainder of the receptor. Such a deletion may have arisen as a result of a microdeletion of genomic DNA or, more likely, as a result of defective splicing.

  3. Loss of exon identity is a common mechanism of human inherited disease

    PubMed Central

    Sterne-Weiler, Timothy; Howard, Jonathan; Mort, Matthew; Cooper, David N.; Sanford, Jeremy R.

    2011-01-01

    It is widely accepted that at least 10% of all mutations causing human inherited disease disrupt splice-site consensus sequences. In contrast to splice-site mutations, the role of auxiliary cis-acting elements such as exonic splicing enhancers (ESE) and exonic splicing silencers (ESS) in human inherited disease is still poorly understood. Here we use a top-down approach to determine rates of loss or gain of known human exonic splicing regulatory (ESR) sequences associated with either disease-causing mutations or putatively neutral single nucleotide polymorphisms (SNPs). We observe significant enrichment toward loss of ESEs and gain of ESSs among inherited disease-causing variants relative to neutral polymorphisms, indicating that exon skipping may play a prominent role in aberrant gene regulation. Both computational and biochemical approaches underscore the relevance of exonic splicing enhancer loss and silencer gain in inherited disease. Additionally, we provide direct evidence that both SRp20 (SRSF3) and possibly PTB (PTBP1) are involved in the function of a splicing silencer that is created de novo by a total of 83 different inherited disease mutations in 67 different disease genes. Taken together, we find that ∼25% (7154/27,681) of known mis-sense and nonsense disease-causing mutations alter functional splicing signals within exons, suggesting a much more widespread role for aberrant mRNA processing in causing human inherited disease than has hitherto been appreciated. PMID:21750108

  4. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

    PubMed Central

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin’ichi; Tsukahara, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing. PMID:27754374

  5. A global regulatory mechanism for activating an exon network required for neurogenesis.

    PubMed

    Raj, Bushra; Irimia, Manuel; Braunschweig, Ulrich; Sterne-Weiler, Timothy; O'Hanlon, Dave; Lin, Zhen-Yuan; Chen, Ginny I; Easton, Laura E; Ule, Jernej; Gingras, Anne-Claude; Eyras, Eduardo; Blencowe, Benjamin J

    2014-10-01

    The vertebrate and neural-specific Ser/Arg (SR)-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells while weakening 3' splice site recognition and contributing to exon skipping in nonneural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis. PMID:25219497

  6. Menzerath-Altmann law in mammalian exons reflects the dynamics of gene structure evolution.

    PubMed

    Nikolaou, Christoforos

    2014-12-01

    Genomic sequences exhibit self-organization properties at various hierarchical levels. One such is the gene structure of higher eukaryotes with its complex exon/intron arrangement. Exon sizes and exon numbers in genes have been shown to conform to a law derived from statistical linguistics and formulated by Menzerath and Altmann, according to which the mean size of the constituents of an entity is inversely related to the number of these constituents. We herein perform a detailed analysis of this property in the complete exon set of the mouse genome in correlation to the sequence conservation of each exon and the transcriptional complexity of each gene locus. We show that extensive linear fits, representative of accordance to Menzerath-Altmann law are restricted to a particular subset of genes that are formed by exons under low or intermediate sequence constraints and have a small number of alternative transcripts. Based on this observation we propose a hypothesis for the law of Menzerath-Altmann in mammalian genes being predominantly due to genes that are more versatile in function and thus, more prone to undergo changes in their structure. To this end we demonstrate one test case where gene categories of different functionality also show differences in the extent of conformity to Menzerath-Altmann law.

  7. A/G Gln20Arg (exon 1) and G/A Val156Met (exon 5) polymorphisms of the human orosomucoid 1 gene in Mexico.

    PubMed

    García-Ortiz, L; Vargas-Alarcón, G; Fragoso, J M; Granados, J; Maldonado Noriega, L; Navas Pérez, A; Huerta Reyes, E; Zenteno-Ruiz, J C; Martínez-Cordero, E

    2008-01-08

    The human orosomucoid 1 gene (ORM1) codes an alpha-1-acid glycoprotein that has been classified as an acute-phase reactive protein, and a major drug-binding serum component, as well as an immunomodulatory protein with genetic polymorphisms. Evaluation of ORM variation through isoelectric focusing and immunobloting has revealed a world-wide distribution of the ORM1 F and ORM1 S alleles. We evaluated and examined the genetic characteristics of two Mexican populations that have different anthropological and cultural antecedents, examining two ORM1 genotypes (exon 1 - A/G (Gln20Arg) and exon 5 G/A (Val156Met)) in 145 individuals, using nested polymerase chain reaction, sequencing, and restricted fragment length polymorphism. Mexican Mestizos had higher frequencies of the exon 1 A allele (P = 0.020) and AA genotype (P = 0.018) and lower frequency of the G allele (P = 0.020) when compared to Teenek Amerindians. When we examined exon 5 G/A (Val156Met) polymorphisms, we found significantly higher frequencies of the G allele (P = 0.0007) and the GG genotype (P = 0.0003) in the Mexican Mestizo population. The Teenek population had a significantly higher frequency of the A allele than has been reported for Chinese and African (P < 0.05) populations, and the G/A genotype was more frequently found in this Mexican population than in Chinese, African and European populations (P < 0.05).

  8. JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

    PubMed Central

    Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; O'Brien, Susan; Giles, Francis; Bruey, Jean Marie; Albitar, Maher

    2010-01-01

    Background The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing. Methodology/Principal Findings We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects. Conclusions/Significance These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for

  9. Homozygous microdeletion of exon 5 in ZNF277 in a girl with specific language impairment.

    PubMed

    Ceroni, Fabiola; Simpson, Nuala H; Francks, Clyde; Baird, Gillian; Conti-Ramsden, Gina; Clark, Ann; Bolton, Patrick F; Hennessy, Elizabeth R; Donnelly, Peter; Bentley, David R; Martin, Hilary; Parr, Jeremy; Pagnamenta, Alistair T; Maestrini, Elena; Bacchelli, Elena; Fisher, Simon E; Newbury, Dianne F

    2014-10-01

    Specific language impairment (SLI), an unexpected failure to develop appropriate language skills despite adequate non-verbal intelligence, is a heterogeneous multifactorial disorder with a complex genetic basis. We identified a homozygous microdeletion of 21,379 bp in the ZNF277 gene (NM_021994.2), encompassing exon 5, in an individual with severe receptive and expressive language impairment. The microdeletion was not found in the proband's affected sister or her brother who had mild language impairment. However, it was inherited from both parents, each of whom carries a heterozygous microdeletion and has a history of language problems. The microdeletion falls within the AUTS1 locus, a region linked to autistic spectrum disorders (ASDs). Moreover, ZNF277 is adjacent to the DOCK4 and IMMP2L genes, which have been implicated in ASD. We screened for the presence of ZNF277 microdeletions in cohorts of children with SLI or ASD and panels of control subjects. ZNF277 microdeletions were at an increased allelic frequency in SLI probands (1.1%) compared with both ASD family members (0.3%) and independent controls (0.4%). We performed quantitative RT-PCR analyses of the expression of IMMP2L, DOCK4 and ZNF277 in individuals carrying either an IMMP2L_DOCK4 microdeletion or a ZNF277 microdeletion. Although ZNF277 microdeletions reduce the expression of ZNF277, they do not alter the levels of DOCK4 or IMMP2L transcripts. Conversely, IMMP2L_DOCK4 microdeletions do not affect the expression levels of ZNF277. We postulate that ZNF277 microdeletions may contribute to the risk of language impairments in a manner that is independent of the autism risk loci previously described in this region. PMID:24518835

  10. Homozygous microdeletion of exon 5 in ZNF277 in a girl with specific language impairment

    PubMed Central

    Ceroni, Fabiola; Simpson, Nuala H; Francks, Clyde; Baird, Gillian; Conti-Ramsden, Gina; Clark, Ann; Bolton, Patrick F; Hennessy, Elizabeth R; Donnelly, Peter; Bentley, David R; Martin, Hilary; Parr, Jeremy; Pagnamenta, Alistair T; Maestrini, Elena; Bacchelli, Elena; Fisher, Simon E; Newbury, Dianne F

    2014-01-01

    Specific language impairment (SLI), an unexpected failure to develop appropriate language skills despite adequate non-verbal intelligence, is a heterogeneous multifactorial disorder with a complex genetic basis. We identified a homozygous microdeletion of 21,379 bp in the ZNF277 gene (NM_021994.2), encompassing exon 5, in an individual with severe receptive and expressive language impairment. The microdeletion was not found in the proband's affected sister or her brother who had mild language impairment. However, it was inherited from both parents, each of whom carries a heterozygous microdeletion and has a history of language problems. The microdeletion falls within the AUTS1 locus, a region linked to autistic spectrum disorders (ASDs). Moreover, ZNF277 is adjacent to the DOCK4 and IMMP2L genes, which have been implicated in ASD. We screened for the presence of ZNF277 microdeletions in cohorts of children with SLI or ASD and panels of control subjects. ZNF277 microdeletions were at an increased allelic frequency in SLI probands (1.1%) compared with both ASD family members (0.3%) and independent controls (0.4%). We performed quantitative RT-PCR analyses of the expression of IMMP2L, DOCK4 and ZNF277 in individuals carrying either an IMMP2L_DOCK4 microdeletion or a ZNF277 microdeletion. Although ZNF277 microdeletions reduce the expression of ZNF277, they do not alter the levels of DOCK4 or IMMP2L transcripts. Conversely, IMMP2L_DOCK4 microdeletions do not affect the expression levels of ZNF277. We postulate that ZNF277 microdeletions may contribute to the risk of language impairments in a manner that is independent of the autism risk loci previously described in this region. PMID:24518835

  11. Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition.

    PubMed Central

    Lavenu, A; Pistoi, S; Pournin, S; Babinet, C; Morello, D

    1995-01-01

    In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2Kb class I regulatory sequences, we have shown that the 5' and the 3' noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3' untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation. PMID:7623834

  12. Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors

    PubMed Central

    Short, Stephen; Peterkin, Tessa; Guille, Matthew; Patient, Roger; Sharpe, Colin

    2015-01-01

    Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus, alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment. PMID:26289800

  13. Genetic variation and balancing selection at MHC class II exon 2 in cultured stocks and wild populations of orange-spotted grouper (Epinephelus coioides).

    PubMed

    Meng, Z N; Yang, S; Fan, B; Wang, L; Lin, H R

    2012-11-12

    Major histocompatibility complex (MHC) molecules play vital roles in triggering adaptive immune responses and are considered the most variable molecules in vertebrates. Recently, many studies have focused on the polymorphism and evolution mode of MHC in both model and non-model organisms. Here, we analyzed the MHC class II exon 2-encoding β chain in comparison with the mitochondrial Cytb gene and our previously published microsatellite data set in three cultured stocks and four wild populations of the orange-spotted grouper (Epinephelus coioides) in order to investigate its genetic variation and mechanism of evolution. We detected one to four alleles in one individual, suggesting that at least two loci exist in the orange-spotted grouper, as well as a particularly high level of allelic diversity at the MHC loci. Furthermore, the cultured stocks exhibited reduced allelic diversity compared to the wild counterparts. We found evidence of balancing selection at MHC class II exon 2, and codon sites under positive selection were largely correspondent to the protein-binding region. In addition, MHC class II exon 2 revealed significant differences between population differentiation patterns from the neutral mitochondrial Cytb and microsatellites, which may indicate local adaptation at MHC loci in orange-spotted grouper originating from the South China Sea and Southeast Asia.

  14. Screening Duchenne and Becker muscular dystrophy patients for deletions in 30 exons of the dystrophin gene by three-multiplex PCR

    SciTech Connect

    Risch, N. )

    1992-09-01

    Deletion mutations of the dystrophin gene may cause either the severe Duchenne muscular dystrophy (DMD) or the milder, allelic Becker muscular dystrophy (BMD) and are clustered in two high-frequency-deletion regions (HFDRs) located, respectively, 500 kb and 1,200 kb downstream from the 5[prime] end of the gene. Three PCR reactions described allowed the analysis of a total of 30 exons and led, to the identification of three additional deletions involving the following exons: (a) 42 only, (b) 28-42, and (c) 16 only, none of which were detected with the two original multiplex reactions. Therefore, the three modified multiplexes detected 95 of the 96 deletions identified among the 152 patients studied so far by using Southern analysis and cDNA probes. The only deletion that remained undetected with this system involves exons 22-25 and generates the junction fragment described elsewhere. The percentage of deletion mutations among DMS/BMD patients amounts to 63%, which is in agreement with similar estimates from other laboratories. When field-inversion gel electrophoresis is coupled to Southern analysis, the detection rate of deletion and duplication mutations reaches 65%.

  15. A Brassica exon array for whole-transcript gene expression profiling.

    PubMed

    Love, Christopher G; Graham, Neil S; O Lochlainn, Seosamh; Bowen, Helen C; May, Sean T; White, Philip J; Broadley, Martin R; Hammond, John P; King, Graham J

    2010-01-01

    Affymetrix GeneChip® arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip® arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip® arrays whose probes are localised primarily in 3' exons. Plant whole-transcript (WT) GeneChip® arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip® Brassica Exon 1.0 ST Array is a 5 µM feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5), with ≤98% sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18), and categorisation by Gene Ontologies (GO) based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.

  16. Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast.

    PubMed

    Bitton, Danny A; Atkinson, Sophie R; Rallis, Charalampos; Smith, Graeme C; Ellis, David A; Chen, Yuan Y C; Malecki, Michal; Codlin, Sandra; Lemay, Jean-François; Cotobal, Cristina; Bachand, François; Marguerat, Samuel; Mata, Juan; Bähler, Jürg

    2015-06-01

    Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5'-3' exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼ 0.24% in wild type and ∼ 1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance.

  17. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

    PubMed Central

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P3H4P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P3H4P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C. PMID:27213357

  18. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis.

    PubMed

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1-2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7-8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P₃H₄P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P₃H₄P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P₃H₄P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5-11.0 and temperature range of 20-40 °C. PMID:27213357

  19. Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast

    PubMed Central

    Bitton, Danny A.; Atkinson, Sophie R.; Rallis, Charalampos; Smith, Graeme C.; Ellis, David A.; Chen, Yuan Y.C.; Malecki, Michal; Codlin, Sandra; Lemay, Jean-François; Cotobal, Cristina; Bachand, François; Marguerat, Samuel; Mata, Juan; Bähler, Jürg

    2015-01-01

    Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5′-3′ exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼0.24% in wild type and ∼1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance. PMID:25883323

  20. Breed-Dependent Transcriptional Regulation of 5′-Untranslated GR (NR3C1) Exon 1 mRNA Variants in the Liver of Newborn Piglets

    PubMed Central

    Yang, Xiaojing; Ni, Yingdong; Cong, Rihua; Soloway, Paul D.; Zhao, Ruqian

    2012-01-01

    Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P<0.05) higher in the liver of newborn piglets of Large White (LW) compared with Erhualian, a Chinese indigenous breed. Overall level of CpG methylation in the region flanking exons 1–4 and 1–5 did not show breed difference. However, nuclear content of Sp1, p-CREB and GR in the liver was significantly (P<0.05) higher in LW piglets, associated with enhanced binding of p-CREB, and higher level of histone H3 acetylation in 1–4 and 1–5 promoters. In contrast, GR binding to promoters of exons 1–4 and 1–5 was significantly diminished in LW piglets, implicating the presence of negative GREs. These results indicate that the difference in the hepatic expression of GR transcript variants between two breeds of pigs is determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3 acetylation to the promoters. PMID:22792317

  1. Breed-dependent transcriptional regulation of 5'-untranslated GR (NR3C1) exon 1 mRNA variants in the liver of newborn piglets.

    PubMed

    Zou, Huafeng; Li, Runsheng; Jia, Yimin; Yang, Xiaojing; Ni, Yingdong; Cong, Rihua; Soloway, Paul D; Zhao, Ruqian

    2012-01-01

    Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5'-untranslated exons 1-4, 1-5, 1-6, 1-7, 1-8, 1-9,10 and 1-11. Among all these mRNA variants, exons 1-4 and 1-5, as well as the total GR were expressed significantly (P<0.05) higher in the liver of newborn piglets of Large White (LW) compared with Erhualian, a Chinese indigenous breed. Overall level of CpG methylation in the region flanking exons 1-4 and 1-5 did not show breed difference. However, nuclear content of Sp1, p-CREB and GR in the liver was significantly (P<0.05) higher in LW piglets, associated with enhanced binding of p-CREB, and higher level of histone H3 acetylation in 1-4 and 1-5 promoters. In contrast, GR binding to promoters of exons 1-4 and 1-5 was significantly diminished in LW piglets, implicating the presence of negative GREs. These results indicate that the difference in the hepatic expression of GR transcript variants between two breeds of pigs is determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3 acetylation to the promoters.

  2. Effect on HIV-1 Gene Expression, Tat-Vpr Interaction and Cell Apoptosis by Natural Variants of HIV-1 Tat Exon 1 and Vpr from Northern India

    PubMed Central

    Lata, Sneh; Ronsard, Larance; Sood, Vikas; Dar, Sajad A.; Ramachandran, Vishnampettai G.; Das, Shukla; Banerjea, Akhil C.

    2013-01-01

    Background Since HIV-1 Tat and Vpr genes are involved in promoter transactivation, apoptosis, etc, we carried out studies to find out nature and extent of natural variation in the two genes from seropositive patients from Northern India and determined their functional implications. Methods HIV-1 tat exon 1 and vpr were amplified from the genomic DNA isolated from the blood of HIV-1 infected individuals using specific primers by Polymerase Chain reaction (PCR) and subjected to extensive genetic analysis (CLUSTAL W, Simplot etc). Their expression was monitored by generating myc fusion clones. Tat exon 1 and Vpr variants were co-transfected with the reporter gene construct (LTR-luc) and their transactivation potential was monitored by measuring luciferase activity. Apoptosis and cell cycle analysis was done by Propidium Iodide (PI) staining followed by FACS. Results Exon 1 of tat was amplified from 21 samples and vpr was amplified from 16 samples. One of the Tat exon 1 variants showed phylogenetic relatedness to subtype B & C and turned out to be a unique recombinant. Two of the Vpr variants were B/C/D recombinants. These natural variations were found to have no impact on the stability of Tat and Vpr. These variants differed in their ability to transactivate B LTR and C LTR promoters. B/C recombinant Tat showed better co-operative interaction with Vpr. B/C/D recombination in Vpr was found to have no effect on its co-operativity with Tat. Recombinant Tat (B/C) induced more apoptosis than wild type B and C Tat. The B/C/D recombination in Vpr did not affect its G2 arrest induction potential but reduced its apoptosis induction ability. Conclusions Extensive sequence and region-specific variations were observed in Tat and Vpr genes from HIV-1 infected individuals from Northern India. These variations have functional implications & therefore important for the pathogenicity of virus. PMID:24367500

  3. Identification of Promotor and Exonic Variations, and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia

    PubMed Central

    Kar, Anjana; Munjal, Sachin Dev; Sarangi, Aditya N.; Dalal, Ashwin; Aggarwal, Rakesh

    2015-01-01

    Background Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)7TAA nucleotide sequence instead of the usual A(TA)6TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India. Methods DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1. Results Of 71 subjects with UH (68 male; median age [range], 26 [16–63] years; serum bilirubin 56 [26–219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)7TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein. Conclusion Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)7TAA

  4. Developing Exon-Primed Intron-Crossing (EPIC) markers for population genetic studies in three Aedes disease vectors.

    PubMed

    White, Vanessa Linley; Endersby, Nancy Margaret; Chan, Janice; Hoffmann, Ary Anthony; Weeks, Andrew Raymond

    2015-03-01

    Aedes aegypti, Aedes notoscriptus, and Aedes albopictus are important vectors of many arboviruses implicated in human disease such as dengue fever. Genetic markers applied across vector species can provide important information on population structure, gene flow, insecticide resistance, and taxonomy, however, robust microsatellite markers have proven difficult to develop in these species and mosquitoes generally. Here we consider the utility and transferability of 15 Ribosome protein (Rp) Exon-Primed Intron-Crossing (EPIC) markers for population genetic studies in these 3 Aedes species. Rp EPIC markers designed for Ae. aegypti also successfully amplified populations of the sister species, Ae. albopictus, as well as the distantly related species, Ae. notoscriptus. High SNP and good indel diversity in sequenced alleles plus support for amplification of the same regions across populations and species were additional benefits of these markers. These findings point to the general value of EPIC markers in mosquito population studies.

  5. Imprinting mutations in Angelman syndrome detected by Southern blotting using a probe containing exon {alpha} of SNRPN

    SciTech Connect

    Beuten, J.; Sutcliffe, J.S.; Nakao, M.

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy (UPD), or other mutations. The SNRPN gene maps in this region, is paternally expressed, and is a candidate gene for PWS. Southern blotting using methylation-sensitive enzymes and a genomic DNA probe from the CpG island containing exon {alpha} of the SNRPN gene reveals methylation specific for the maternal allele. In cases of the usual deletions or UPD, the probe detects absence of an unmethylated allele in PWS and absence of a methylated allele in AS. We have analyzed 21 nondeletion/nonUPD AS patients with this probe and found evidence for an imprinting mutation (absence of a methylated allele) in 3 patients. Southern blotting with methylation-sensitive enzymes using the exon {alpha} probe, like use of the PW71 probe, should detect abnormalities in all known PWS cases and in 3 of the 4 forms of AS: deletion, UPD and imprinting mutations. This analysis provides a valuable diagnostic approach for PWS and AS. In efforts to localize the imprinting mutations in AS, one patient was found with failure to inherit a dinucleotide repeat polymorphism near probe 189-1 (D15S13). Analysis of this locus in AS families and CEPH families demonstrates a polymorphism that impairs amplification and a different polymorphism involving absence of hybridization to the 189-1 probe. The functional significance, if any, of deletion of the 189-1 region is unclear.

  6. Mutation analysis of exon1 of bone morphogenetic protein-15 gene in Iranian patients with polycystic ovarian syndrome

    PubMed Central

    Mehdizadeh, Anahita; Sheikhha, Mohammad Hasan; Kalantar, Seyed Mehdi; Aali, Bibi Shahnaz; Ghanei, Azam

    2016-01-01

    Background: With the prevalence of 6-10%, polycystic ovarian syndrome (PCOS) is considered the most common endocrinological disorder affecting women in their reproductive age. It has been suggested that genetic factors participate in the development of PCOS. Follicular development has been considered as one of the impaired processes in PCOS. Bone morphogenetic protein-15 (BMP-15) gene is a candidate gene in follicular development and its variants may play role in pathogenesis of PCOS. Objective: To investigate whether BMP-15 gene mutations are present in Iranian women with PCOS. Materials and Methods: In this cross-sectional study 5 ml venous blood samples was taken from 70 PCOS women referring to Afzalipour Hospital, Kerman University of Medical Sciences, Kerman, Iran, between January to December 2014. Genomic DNA was extracted from the blood sample by salting out method. Then a set of PCR reactions for exon1 of BMP-15 gene was performed using specific primers followed by genotyping with direct sequencing. Results: Two different polymorphisms were found in the gene under study. In total 20 patients (28.6%) were heterozygote (C/G), and 2 patients (2.86%) were homozygous (G/G) for c.-9C>G in 5´UTR promoter region of BMP-15 gene (rs3810682). In addition, in the coding region of exon1, three patients (4.3%) were heterozygote (G/A) for c.A308G (rs41308602). Two PCOS patients (2.86%) appeared to have both c.-9C>G (C/G) and c.A308G (G/A) variants simultaneously. Conclusion: Our research detected two polymorphisms of BMP-15 gene among PCOS patients, indicating that even though it cannot be concluded that variants of BMP-15 gene are the principal cause of polycystic ovarian syndrome; they could be involved in pathogenic process in development of PCOS.

  7. Altered Striatal Synaptic Function and Abnormal Behaviour in Shank3 Exon4-9 Deletion Mouse Model of Autism.

    PubMed

    Jaramillo, Thomas C; Speed, Haley E; Xuan, Zhong; Reimers, Jeremy M; Liu, Shunan; Powell, Craig M

    2016-03-01

    Shank3 is a multi-domain, synaptic scaffolding protein that organizes proteins in the postsynaptic density of excitatory synapses. Clinical studies suggest that ∼ 0.5% of autism spectrum disorder (ASD) cases may involve SHANK3 mutation/deletion. Patients with SHANK3 mutations exhibit deficits in cognition along with delayed/impaired speech/language and repetitive and obsessive/compulsive-like (OCD-like) behaviors. To examine how mutation/deletion of SHANK3 might alter brain function leading to ASD, we have independently created mice with deletion of Shank3 exons 4-9, a region implicated in ASD patients. We find that homozygous deletion of exons 4-9 (Shank3(e4-9) KO) results in loss of the two highest molecular weight isoforms of Shank3 and a significant reduction in other isoforms. Behaviorally, both Shank3(e4-9) heterozygous (HET) and Shank3(e4-9) KO mice display increased repetitive grooming, deficits in novel and spatial object recognition learning and memory, and abnormal ultrasonic vocalizations. Shank3(e4-9) KO mice also display abnormal social interaction when paired with one another. Analysis of synaptosome fractions from striata of Shank3(e4-9) KO mice reveals decreased Homer1b/c, GluA2, and GluA3 expression. Both Shank3(e4-9) HET and KO demonstrated a significant reduction in NMDA/AMPA ratio at excitatory synapses onto striatal medium spiny neurons. Furthermore, Shank3(e4-9) KO mice displayed reduced hippocampal LTP despite normal baseline synaptic transmission. Collectively these behavioral, biochemical and physiological changes suggest Shank3 isoforms have region-specific roles in regulation of AMPAR subunit localization and NMDAR function in the Shank3(e4-9) mutant mouse model of autism. PMID:26559786

  8. Mutation analysis of exon1 of bone morphogenetic protein-15 gene in Iranian patients with polycystic ovarian syndrome

    PubMed Central

    Mehdizadeh, Anahita; Sheikhha, Mohammad Hasan; Kalantar, Seyed Mehdi; Aali, Bibi Shahnaz; Ghanei, Azam

    2016-01-01

    Background: With the prevalence of 6-10%, polycystic ovarian syndrome (PCOS) is considered the most common endocrinological disorder affecting women in their reproductive age. It has been suggested that genetic factors participate in the development of PCOS. Follicular development has been considered as one of the impaired processes in PCOS. Bone morphogenetic protein-15 (BMP-15) gene is a candidate gene in follicular development and its variants may play role in pathogenesis of PCOS. Objective: To investigate whether BMP-15 gene mutations are present in Iranian women with PCOS. Materials and Methods: In this cross-sectional study 5 ml venous blood samples was taken from 70 PCOS women referring to Afzalipour Hospital, Kerman University of Medical Sciences, Kerman, Iran, between January to December 2014. Genomic DNA was extracted from the blood sample by salting out method. Then a set of PCR reactions for exon1 of BMP-15 gene was performed using specific primers followed by genotyping with direct sequencing. Results: Two different polymorphisms were found in the gene under study. In total 20 patients (28.6%) were heterozygote (C/G), and 2 patients (2.86%) were homozygous (G/G) for c.-9C>G in 5´UTR promoter region of BMP-15 gene (rs3810682). In addition, in the coding region of exon1, three patients (4.3%) were heterozygote (G/A) for c.A308G (rs41308602). Two PCOS patients (2.86%) appeared to have both c.-9C>G (C/G) and c.A308G (G/A) variants simultaneously. Conclusion: Our research detected two polymorphisms of BMP-15 gene among PCOS patients, indicating that even though it cannot be concluded that variants of BMP-15 gene are the principal cause of polycystic ovarian syndrome; they could be involved in pathogenic process in development of PCOS. PMID:27679828

  9. Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

    PubMed

    Rutten, Julie W; Dauwerse, Hans G; Peters, Dorien J M; Goldfarb, Andrew; Venselaar, Hanka; Haffner, Christof; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M; Lesnik Oberstein, Saskia A J

    2016-04-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations

  10. Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

    PubMed Central

    Dewaele, Michael; Tabaglio, Tommaso; Willekens, Karen; Bezzi, Marco; Teo, Shun Xie; Low, Diana H.P.; Koh, Cheryl M.; Rambow, Florian; Fiers, Mark; Rogiers, Aljosja; Radaelli, Enrico; Al-Haddawi, Muthafar; Tan, Soo Yong; Hermans, Els; Amant, Frederic; Yan, Hualong; Lakshmanan, Manikandan; Koumar, Ratnacaram Chandrahas; Lim, Soon Thye; Derheimer, Frederick A.; Campbell, Robert M.; Bonday, Zahid; Tergaonkar, Vinay; Shackleton, Mark; Blattner, Christine; Marine, Jean-Christophe; Guccione, Ernesto

    2015-01-01

    MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient–derived xenograft (PDX) mouse models, antisense oligonucleotide–mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target. PMID:26595814

  11. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    PubMed Central

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  12. iGEMS: an integrated model for identification of alternative exon usage events

    PubMed Central

    Sood, Sanjana; Szkop, Krzysztof J.; Nakhuda, Asif; Gallagher, Iain J.; Murie, Carl; Brogan, Robert J.; Kaprio, Jaakko; Kainulainen, Heikki; Atherton, Philip J.; Kujala, Urho M.; Gustafsson, Thomas; Larsson, Ola; Timmons, James A.

    2016-01-01

    DNA microarrays and RNAseq are complementary methods for studying RNA molecules. Current computational methods to determine alternative exon usage (AEU) using such data require impractical visual inspection and still yield high false-positive rates. Integrated Gene and Exon Model of Splicing (iGEMS) adapts a gene-level residuals model with a gene size adjusted false discovery rate and exon-level analysis to circumvent these limitations. iGEMS was applied to two new DNA microarray datasets, including the high coverage Human Transcriptome Arrays 2.0 and performance was validated using RT-qPCR. First, AEU was studied in adipocytes treated with (n = 9) or without (n = 8) the anti-diabetes drug, rosiglitazone. iGEMS identified 555 genes with AEU, and robust verification by RT-qPCR (∼90%). Second, in a three-way human tissue comparison (muscle, adipose and blood, n = 41) iGEMS identified 4421 genes with at least one AEU event, with excellent RT-qPCR verification (95%, n = 22). Importantly, iGEMS identified a variety of AEU events, including 3′UTR extension, as well as exon inclusion/exclusion impacting on protein kinase and extracellular matrix domains. In conclusion, iGEMS is a robust method for identification of AEU while the variety of exon usage between human tissues is 5–10 times more prevalent than reported by the Genotype-Tissue Expression consortium using RNA sequencing. PMID:27095197

  13. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy.

    PubMed

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  14. rMAPS: RNA map analysis and plotting server for alternative exon regulation

    PubMed Central

    Park, Juw Won; Jung, Sungbo; Rouchka, Eric C.; Tseng, Yu-Ting; Xing, Yi

    2016-01-01

    RNA-binding proteins (RBPs) play a critical role in the regulation of alternative splicing (AS), a prevalent mechanism for generating transcriptomic and proteomic diversity in eukaryotic cells. Studies have shown that AS can be regulated by RBPs in a binding-site-position dependent manner. Depending on where RBPs bind, splicing of an alternative exon can be enhanced or suppressed. Therefore, spatial analyses of RBP motifs and binding sites around alternative exons will help elucidate splicing regulation by RBPs. The development of high-throughput sequencing technologies has allowed transcriptome-wide analyses of AS and RBP–RNA interactions. Given a set of differentially regulated alternative exons obtained from RNA sequencing (RNA-seq) experiments, the rMAPS web server (http://rmaps.cecsresearch.org) performs motif analyses of RBPs in the vicinity of alternatively spliced exons and creates RNA maps that depict the spatial patterns of RBP motifs. Similarly, rMAPS can also perform spatial analyses of RBP–RNA binding sites identified by cross-linking immunoprecipitation sequencing (CLIP-seq) experiments. We anticipate rMAPS will be a useful tool for elucidating RBP regulation of alternative exon splicing using high-throughput sequencing data. PMID:27174931

  15. Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

    PubMed Central

    Winnard, A V; Mendell, J R; Prior, T W; Florence, J; Burghes, A H

    1995-01-01

    Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7825572

  16. Exonic splicing signals impose constraints upon the evolution of enzymatic activity.

    PubMed

    Falanga, Alessia; Stojanović, Ozren; Kiffer-Moreira, Tina; Pinto, Sofia; Millán, José Luis; Vlahoviček, Kristian; Baralle, Marco

    2014-05-01

    Exon splicing enhancers (ESEs) overlap with amino acid coding sequences implying a dual evolutionary selective pressure. In this study, we map ESEs in the placental alkaline phosphatase gene (ALPP), absent in the corresponding exon of the ancestral tissue-non-specific alkaline phosphatase gene (ALPL). The ESEs are associated with amino acid differences between the transcripts in an area otherwise conserved. We switched out the ALPP ESEs sequences with the sequence from the related ALPL, introducing the associated amino acid changes. The resulting enzymes, produced by cDNA expression, showed different kinetic characteristics than ALPL and ALPP. In the organism, this enzyme will never be subjected to selection because gene splicing analysis shows exon skipping due to loss of the ESE. Our data prove that ESEs restrict the evolution of enzymatic activity. Thus, suboptimal proteins may exist in scenarios when coding nucleotide changes and consequent amino acid variation cannot be reconciled with the splicing function. PMID:24692663

  17. The stop mutation R553X in the CFTR gene results in exon skipping

    SciTech Connect

    Hull, J.; Shackleton, S.; Harris, A. )

    1994-01-15

    Stop or nonsense mutations are known to disrupt gene function in a number of different ways. The authors have studied the effects of the stop mutation R553X in exon 11 of the CFTR gene by analyzing mRNA extracted from nasal epithelial cells harvested from patients with cystic fibrosis. Four patients who were compound heterozygotes for the R553X mutation were studied. Ten non-CF control subjects were also studied. In all four patients, full-length CFTR mRNA was identified, but only a very small proportion of this was derived from the R553X allele. A smaller transcript, lacking exon 11, was also seen in the R553X patients but not in the controls. Most of this transcript was derived from the R553X allele. These results suggest that the R553X mutation results in skipping of the exon in which it is located. 14 refs., 3 figs.

  18. Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 3' splice site of intron 1)

    PubMed

    Aldred, A R; Prapunpoj, P; Schreiber, G

    1997-06-01

    Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid sequence of kangaroo transthyretin, together with data obtained previously for transthyretins from other vertebrate species [Duan, W., Richardson, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J., Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396-406], showed that the N-terminus is the region which changes most distinctly during evolution. It has been shown for human, mouse and rat transthyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was amplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wallaby, Eastern Grey Kangaroo, Stripe-faced Dunnart, Short-tailed Grey Opossum and White Leghorn Chicken. The splice sites at both ends of intron 1 were identified by comparison with the cDNA sequences. The obtained data suggest that the N-termini of transthyretin evolved by successive shifts of the 3' splice site of intron 1 in the 3' direction, resulting in successive shortening of the 5' end of exon 2. At the protein level, this resulted in a shorter and more hydrophilic N-terminal region of transthyretin. Successive shifts in splice sites may be an evolutionary mechanism of general importance, since they can lead to stepwise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection.

  19. Structural organization of the human S-antigen gene. cDNA, amino acid, intron, exon, promoter, in vitro transcription, retina, and pineal gland.

    PubMed

    Yamaki, K; Tsuda, M; Kikuchi, T; Chen, K H; Huang, K P; Shinohara, T

    1990-12-01

    S-Antigen (S-Ag) is a major soluble photoreceptor protein involved in the visual transduction cascade. Several S-Ag cDNAs and a gene coding for human S-Ag were isolated from cDNA and gene libraries. The gene sequences of the coding, noncoding, and 5'-flanking regions of the gene were determined. The S-Ag gene was approximately 50 kbp (kilobase pairs) in length and contained 16 exons and 15 introns. The length of most exons was less than 100 base pairs (bp) and the smallest one was only 10 bp. In contrast, the length of most introns was larger than 2 kbp, and the gene comprised 97% intron and 3% exon. The splice sites for donor and acceptor were in good agreement with the GT/AG rule. The S-Ag protein of 403 amino acid residues was translated from a mRNA of 1.9 kbp, and the mRNA was transcribed from a gene of 50 kbp. The 5'-flanking region of the gene, approximately 1.1 kbp long, had no known regulatory elements for transcription such as TATA, GC, and CCAAT boxes. Interestingly, the 5'-flanking region had promoter activity in an in vitro transcription assay using a nuclear extract of rat brain. A major transcription start site was found at 387 bp upstream from the translation start site ATG. Our results indicate that the sequence of S-Ag promoter differs from other known promoters and may, perhaps, be specific for photoreceptor rod cells and pinealocytes.

  20. Neonatal Marfan syndrome caused by an exon 25 mutation of the fibrillin-1 gene.

    PubMed

    Elçioglu, N H; Akalin, F; Elçioglu, M; Comeglio, P; Child, A H

    2004-01-01

    Neonatal Marfan syndrome caused by an exon 25 mutation of the Fibrillin-1 gene: We describe a male infant with severe arachnodactyly, hypermobility of the fingers, flexion contractures of elbows, wrists, hips, and knees, microretrognathia, crumpled ears, rockerbottom feet, loose redundant skin, and lens dislocations. Cardiac valve insufficiency and aortic dilatation resulted in cardiac failure, decompensated with digitalisation and death occurred at the age of 4 months. This case represents the severe end of the clinical spectrum of Marfan syndrome, namely neonatal Marfan syndrome. Molecular diagnostic analyses confirmed a de novo exon 25 mutation in the FBN1 gene. PMID:15287423

  1. In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes

    PubMed Central

    SINGH, NATALIA N.; ANDROPHY, ELLIOT J.; SINGH, RAVINDRA N.

    2004-01-01

    Humans have two near identical copies of the survival of motor neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to an inhibitory mutation at position 6 (C6U mutation in transcript) of exon 7. We have recently shown that C6U creates an extended inhibitory context (Exinct) that causes skipping of exon 7 in SMN2. Previous studies have shown that an exonic splicing enhancer associated with Tra2 (Tra2-ESE) is required for exon 7 inclusion in both SMN1 and SMN2. Here we describe the method of in vivo selection that determined the position-specific role of wild-type nucleotides within the entire exon 7. Our results confirmed the existence of Exinct and revealed the presence of an additional inhibitory tract (3′-Cluster) near the 3′-end of exon 7. We also demonstrate that a single nucleotide substitution at the last position of exon 7 improves the 5′ splice site (ss) such that the presence of inhibitory elements (Exinct as well as the 3′-Cluster) and the absence of Tra2-ESE no longer determined exon 7 usage. Our results suggest that the evolutionary conserved weak 5′ ss may serve as a mechanism to regulate exon 7 splicing under different physiological contexts. This is the first report in which a functional selection method has been applied to analyze the entire exon. This method offers unparallel advantage for determining the relative strength of splice sites, as well as for identifying the novel exonic cis-elements. PMID:15272122

  2. hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure.

    PubMed Central

    Damgaard, Christian Kroun; Tange, Thomas Ostergaard; Kjems, Jørgen

    2002-01-01

    The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point. PMID:12458794

  3. Expression and New Exon Mutations of the Human Beta Defensins and Their Association on Colon Cancer Development

    PubMed Central

    Semlali, Abdelhabib; Al Amri, Abdullah; Azzi, Arezki; Al Shahrani, Omair; Arafah, Maha; Kohailan, Muhammad; Aljebreen, Abdulrahman M.; alharbi, Othman; Almadi, Majid A.; Azzam, Nahla Ali; Parine, Narasimha Reddy; Rouabhia, Mahmoud; Alanazi, Mohammad S.

    2015-01-01

    The development of cancer involves genetic predisposition and a variety of environmental exposures. Genome-wide linkage analyses provide evidence for the significant linkage of many diseases to susceptibility loci on chromosome 8p23, the location of the human defensin gene cluster. Human β-defensins (hBDs) are important molecules of innate immunity. This study was designed to analyze the expression and genetic variations in hBDs (hBD-1, hBD-2, hBD-3 and hBD-4) and their putative association with colon cancer. hBD gene expression and relative protein expression were evaluated by Real-Time polymerase chain reaction (qPCR) and immunohistochemistry, respectively, from 40 normal patients and 40 age-matched patients with colon cancer in Saudi Arabia. In addition, hBD polymorphisms were genotyped by exon sequencing and by promoter methylation. hBD-1, hBD-2, hBD-3 and hBD-4 basal messenger RNA expression was significantly lower in tumor tissues compared with normal tissues. Several insertion mutations were detected in different exons of the analyzed hBDs. However, no methylation in any hBDs promoters was detected because of the limited number of CpG islands in these regions. We demonstrated for the first time a link between hBD expression and colon cancer. This suggests that there is a significant link between innate immunity deregulation through disruption of cationic peptides (hBDs) and the potential development of colon cancer. PMID:26038828

  4. Molecular basis of two-exon skipping (exons 12 and 13) by c.1248+5g>a in OXCT1 gene: study on intermediates of OXCT1 transcripts in fibroblasts.

    PubMed

    Hori, Tomohiro; Fukao, Toshiyuki; Murase, Keiko; Sakaguchi, Naomi; Harding, Cary O; Kondo, Naomi

    2013-03-01

    The molecular basis of simultaneous two-exon skipping induced by a splice-site mutation has yet to be completely explained. The splice donor site mutation c.1248+5g>a (IVS13) of the OXCT1 gene resulted predominantly in skipping of exons 12 and 13 in fibroblasts from a patient (GS23) with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency. We compared heteronuclear RNA (hnRNA) intermediates between controls' and GS23's fibroblasts. Our strategy was to use RT-PCR of hnRNA to detect the presence or absence of spliced exon clusters in RNA intermediates (SECRIs) comprising sequential exons. Our initial hypothesis was that a SECRI comprising exons 12 and 13 was formed first followed by skipping of this SECRI in GS23 cells. However, such a pathway was revealed to be not a major one. Hence, we compared the intron removal of SCOT transcript between controls and GS23. In controls, intron 11 was the last intron to be spliced and the removal of intron 12 was also rather slow and occurred after the removal of intron 13 in a major pathway. However, the mutation in GS23 cells resulted in retention of intron 13, thus causing the retention of introns 12 and 11. This "splicing paralysis" may be solved by skipping the whole intron 11-exon 12-intron 12-exon 13-mutated intron 13, resulting in skipping of exons 12 and 13. PMID:23281106

  5. Plug-and-play genetic access to drosophila cell types using exchangeable exon cassettes.

    PubMed

    Diao, Fengqiu; Ironfield, Holly; Luan, Haojiang; Diao, Feici; Shropshire, William C; Ewer, John; Marr, Elizabeth; Potter, Christopher J; Landgraf, Matthias; White, Benjamin H

    2015-03-01

    Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of "coding introns" (i.e., introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted "plug-and-play" cassettes (called "Trojan exons") that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette) transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system. PMID:25732830

  6. Improvements to previous algorithms to predict gene structure and isoform concentrations using Affymetrix Exon arrays

    PubMed Central

    2010-01-01

    Background Exon arrays provide a way to measure the expression of different isoforms of genes in an organism. Most of the procedures to deal with these arrays are focused on gene expression or on exon expression. Although the only biological analytes that can be properly assigned a concentration are transcripts, there are very few algorithms that focus on them. The reason is that previously developed summarization methods do not work well if applied to transcripts. In addition, gene structure prediction, i.e., the correspondence between probes and novel isoforms, is a field which is still unexplored. Results We have modified and adapted a previous algorithm to take advantage of the special characteristics of the Affymetrix exon arrays. The structure and concentration of transcripts -some of them possibly unknown- in microarray experiments were predicted using this algorithm. Simulations showed that the suggested modifications improved both specificity (SP) and sensitivity (ST) of the predictions. The algorithm was also applied to different real datasets showing its effectiveness and the concordance with PCR validated results. Conclusions The proposed algorithm shows a substantial improvement in the performance over the previous version. This improvement is mainly due to the exploitation of the redundancy of the Affymetrix exon arrays. An R-Package of SPACE with the updated algorithms have been developed and is freely available. PMID:21110835

  7. Mucopolysaccharidosis IVA: Four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency

    SciTech Connect

    Tomatsu, Shunji; Fukuda, Seiji; Yamagishi, Atsushi

    1996-05-01

    We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations: V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resulted in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation. 48 refs., 5 figs., 1 tab.

  8. Polypurine sequences within a downstream exon function as a splicing enhancer

    SciTech Connect

    Tanaka, Kenji; Watakabe, Akiya; Shimura, Yoshiro

    1994-02-01

    We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin {mu} gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin {mu} gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection. 50 refs., 7 figs., 2 tabs.

  9. Mutations in exon 10 of the RET proto-oncogene in Hirschsprung`s disease

    SciTech Connect

    Attie, T.; Eng, C.; Mulligan, L.M.

    1994-09-01

    Hirschsprung`s disease (HSCR) is a frequent congenital malformation ascribed to the absence of autonomic ganglion cells in the terminal hindgut. Recently, we have identified mutations in the RET proto-oncogene in HSCR families. Mutations of the RET gene have also been reported in multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). While RET mutations in HSCR are scattered on the whole coding sequence, MEN 2A and FMTC mutations are clustered in 5 cystein codons of exons 10 and 11. Here, we report on HSCR families carrying mutations in exon 10 of the RET gene, one of them involving a cystein codon. Germ-line mutations in exon 10 of the RET gene may contribute to either an early development defect (HSCR) or inherited predisposition to cancer (MEN 2A and FMTC), probable depending on the nature and location of the mutation. These data also suggest that HSCR patients with mutations in exon 10 might subsequently prove to be at risk for MEN 2A or FMTC since several MEN 2A/HSCR associations have been reported.

  10. A novel first exon directs hormone-sensitive transcription of the pig prolactin receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endocrine, paracrine, and autocrine prolactin (PRL) acts through its receptor (PRLR) to confer a wide range of biological functions, including its established role during lactation.We have identified a novel first exon of the porcine PRLR that gives rise to three different mRNA transcripts. Transcri...

  11. Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer.

    PubMed

    Sahoo, Bankanidhi; Arduini, Irene; Drombosky, Kenneth W; Kodali, Ravindra; Sanders, Laurie H; Greenamyre, J Timothy; Wetzel, Ronald

    2016-01-01

    Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile

  12. Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer

    PubMed Central

    Sahoo, Bankanidhi; Arduini, Irene; Drombosky, Kenneth W.; Kodali, Ravindra; Sanders, Laurie H.; Greenamyre, J. Timothy; Wetzel, Ronald

    2016-01-01

    Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington’s disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6–9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction

  13. Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells

    SciTech Connect

    Kang, Wen; Mukerjee, Ruma; Gartner, Jared J.; Hatzigeorgiou, Artemis G.; Sandri-Goldin, Rozanne M.; Fraser, Nigel W. . E-mail: nfraser@mail.med.upenn.edu

    2006-12-20

    The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells.

  14. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

    PubMed Central

    Hoop, Cody L.; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M.; Boatz, Jennifer C.; Mandal, Abhishek; Lewandowski, Józef R.; Wetzel, Ronald

    2016-01-01

    Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin–containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand–based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of “intrinsic” polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms. PMID:26831073

  15. NF1 Exon 22 Analysis of Individuals with the Clinical Diagnosis of Neurofibromatosis Type 1

    PubMed Central

    Muram-Zborovski, Talia M.; Vaughn, Cecily P.; Viskochil, David H.; Hanson, Heather; Mao, Rong; Stevenson, David A.

    2010-01-01

    Café-au-lait macules are frequently seen in Ras-MAPK pathway disorders and are a cardinal feature of neurofibromatosis type 1 (NF1). Most NF1 individuals develop age-related tumorigenic manifestations (e.g. neurofibromas), although individuals with a specific 3-bp deletion in exon 22 of NF1 (c.2970_2972delAAT) have an attenuated phenotype with primarily pigmentary manifestations. Previous reports identify this deletion c.2970_2972delAAT in exon 17 of NF1 using NF Consortium nomenclature. For this report, we elected to use standard NCBI nomenclature, which places this identical deletion within exon 22. SPRED1 causes Legius syndrome, which clinically overlaps with this attenuated NF1 phenotype. In an unselected cohort of 150 individuals who fulfilled NIH clinical diagnostic criteria from an NF Clinic and did not have SPRED1 mutations, we sequenced NF1 exon 22 in order to identify children and adolescents with multiple café-au-lait spots who could be projected to have lower likelihood to develop tumors. Two individuals with NF1 exon 22 mutations were identified: an 11-year-old boy with the c.2970_2972delAAT in-frame deletion and a 4-year-old boy with c.2866dupA. The father of the second patient had an attenuated form of NF1 and showed 24% germline mosaicism of the c.2866dupA mutation in whole blood. These individuals emphasize the need for mutation analysis in some individuals with the clinical diagnosis of NF1 who lack the tumorigenic or classic skeletal abnormalities of NF1. Specifically, with the identification of Legius syndrome, the need to recognize the attenuated phenotype of NF1 mosaicism and confirmation by mutation analysis is increasingly important for appropriate medical management and family counseling. PMID:20602485

  16. Aggregation Behavior of Chemically Synthesized, Full-Length Huntingtin Exon1

    PubMed Central

    2015-01-01

    Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington’s disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued. PMID:24921664

  17. Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis.

    PubMed

    Spokony, Rebecca F; Restifo, Linda L

    2007-07-01

    Broad Complex (BRC) is an essential ecdysone-pathway gene required for entry into and progression through metamorphosis in Drosophila melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. These defects are phenocopied by the juvenile hormone mimic methoprene. Four BRC isoforms are produced by alternative splicing of a protein-binding BTB/POZ-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC). Highly conserved orthologs of BTBBRC and all four ZBRC were found among published cDNA sequences or genome databases from Diptera, Lepidoptera, Hymenoptera, and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometabolous orders. Tramtrack subfamily members, abrupt, tramtrack, fruitless, longitudinals lacking (lola), and CG31666 were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, which revealed that the ZBRC clade includes Zabrupt. All four ZBRC domains, including Z4BRC, which has no known essential function, are evolving in a manner consistent with selective constraint. We used transgenic rescue to explore how different BRC isoforms contribute to shared tissue-morphogenesis functions. As predicted from earlier studies, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two different developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. The sequential action of BRC isoforms indicates subfunctionalization of duplicated ZBRC exons even when they contribute to common developmental processes.

  18. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    PubMed Central

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  19. Deletion of SNURF/SNRPN U1B and U1B* upstream exons in a child with developmental delay and excessive weight.

    PubMed

    Koufaris, Costas; Alexandrou, Angelos; Papaevripidou, Ioannis; Alexandrou, Ioanna; Christophidou-Anastasiadou, Violetta; Sismani, Carolina

    2016-09-01

    Prader-Willi syndrome is a rare syndrome characterized by hypotonia, developmental delay and excessive appetite. This syndrome is caused by the loss of function of paternally-expressed genes located in an imprinting centre in 15q11-q13. Here, we report the case of a patient who was referred to us with Prader-Willi syndrome-like symptoms including obesity and developmental delay. Examination of this patient revealed that he was a carrier of a paternally inherited deletion that affected the U1B and U1B* upstream exons of the SNURF-SNRNP gene within the 15q11-q13 imprinted region. Mutations localized within this genomic region have not been previously reported in Prader-Willi syndrome patients. It is possible that disruption of upstream exons of SNURF-SNRNP could contribute to Prader-Willi phenotype by disrupting brain-specific alternative transcripts, although, case reports from further patients with a comparable phenotype are required. PMID:27659333

  20. A novel SMAD family protein, SMAD9 is involved in follicular initiation and changes egg yield of geese via synonymous mutations in exon1 and intron2.

    PubMed

    Xu, Jun; Li, Jun; Wang, Haosen; Wang, Guanglin; Chen, Jie; Huang, Pin; Cheng, Jienan; Gan, Lu; Wang, Zhao; Cai, Yafei

    2015-01-01

    Elevation of egg performance is vital to goose farming. Many poultry scientists are seeking for efficient molecular genetic markers associated with egg yield. In this study, mRNA differential display was adopted to investigate gene expression profiling in the follicular development of goose. For the first time, a novel SMAD family protein SMAD9 (EST CJ111007) was found to be involved in follicular initiation and used to be a candidate gene. Functional regions analysis of Smad9 indicated that SMAD9 protein is highly conserved in MH1 and MH2 domains, and the connection area is highly variable region. 6 pairs of primers (p1-p6) were designed to detect the SNPs of Smad9 by PCR-SSCP method. The results revealed that polymorphisms were discovered in the PCR products amplified with P1 primers in exon1 and P3 primers in intron2. In Smad9 exon1, 5 genotypes were found: FK, FF, JJ, JK and KK, including 2 SNPs: 243 bp G → A, 309 bp T → G, the mutations did not result in amino acid mutations; In intron2, 3 genotypes were found: AA, BB and AB, only 1 SNP (C → T). The annual egg yield of FK genotype geese or allele gene A in intron2 are significantly more than those of other genotypes on the average (p < 0.05). Taken together, it is suggested that Smad9 is a promising candidate gene affecting egg performance in goose.

  1. The assessment of noncoding variant of PPOX gene in variegate porphyria reveals post-transcriptional role of the 5' untranslated exon 1.

    PubMed

    Fiorentino, Valeria; Brancaleoni, Valentina; Granata, Francesca; Graziadei, Giovanna; Di Pierro, Elena

    2016-10-01

    The PPOX gene encodes for the protoporphyrinogen oxidase, which is involved in heme production. The partial deficiency of protoporphyrinogen oxidase causes variegate porphyria. The tissue-specific regulation of other heme biosynthetic enzymes is extensively studied, but the information concerning transcriptional and post-transcriptional regulation of PPOX gene expression is scarcely available. In this study, we characterized functions of three variants identified in the regulatory regions of the PPOX gene, which show a novel role for the 5' untranslated exon 1. Using luciferase assays and RNA analysis, we demonstrated that only c.1-883G>C promoter variant causes a significant loss in the transcriptional activity of PPOX gene whereas c.1-413G>T 5' UTR variant inhibits translation of PPOX mRNA and c.1-176G>A splicing variant causes 4bp deletion in 5' UTR of PPOX mRNA variant 2. These observations indicate that the regulation of PPOX gene expression can also occur through a post-transcriptional modulation of the amount of gene product and that this modulation can be mediated by 5' untranslated exon 1. Moreover this study confirms that these regulatory regions represent an important molecular target for the pathogenesis of variegate porphyria. PMID:27667166

  2. Deletion of the carboxyl-terminal exons of K-sam/FGFR2 by short homology-mediated recombination, generating preferential expression of specific messenger RNAs.

    PubMed

    Ueda, T; Sasaki, H; Kuwahara, Y; Nezu, M; Shibuya, T; Sakamoto, H; Ishii, H; Yanagihara, K; Mafune, K; Makuuchi, M; Terada, M

    1999-12-15

    The K-sam gene was first identified as an amplified gene from human gastric cancer cell line KATOIII, and its product is identical to fibroblast growth factor receptor 2. The K-sam gene is located on human chromosome 10q26 and is preferentially amplified in the poorly differentiated types, especially in the scirrhous type, of gastric cancers. During the course of studies on the structural characterization of the amplification units, we found that the carboxyl-terminal exons of K-sam were deleted in three of four of the scirrhous type of gastric cancer cell lines. These deletions generate preferential expression of mRNAs encoding K-sam proteins lacking the carboxyl-terminal region containing the tyrosine residues at positions 780, 784, and 813. The carboxyl-terminal region has been reported to have a sequence required for the inhibition of NIH3T3 transformation, indicating that cells with amplification of the truncated K-sam gene have a growth advantage during the carcinogenic process for the scirrhous type of gastric cancers. This is the first report showing the deletion of the carboxyl-terminal exons of the receptor-type of the protein tyrosine kinase gene. Sequence analysis of the DNA sequences surrounding the deletion junctions shows the presence of unique sequences and indicates the involvement of short homology-mediated recombination in the generation of these deletions. PMID:10626794

  3. A silent exonic SNP in kdm3a affects nucleic acids structure but does not regulate experimental autoimmune encephalomyelitis.

    PubMed

    Gillett, Alan; Bergman, Petra; Parsa, Roham; Bremges, Andreas; Giegerich, Robert; Jagodic, Maja

    2013-01-01

    Defining genetic variants that predispose for diseases is an important initiative that can improve biological understanding and focus therapeutic development. Genetic mapping in humans and animal models has defined genomic regions controlling a variety of phenotypes known as quantitative trait loci (QTL). Causative disease determinants, including single nucleotide polymorphisms (SNPs), lie within these regions and can often be identified through effects on gene expression. We previously identified a QTL on rat chromosome 4 regulating macrophage phenotypes and immune-mediated diseases including experimental autoimmune encephalomyelitis (EAE). Gene analysis and a literature search identified lysine-specific demethylase 3A (Kdm3a) as a potential regulator of these phenotypes. Genomic sequencing determined only two synonymous SNPs in Kdm3a. The silent synonymous SNP in exon 15 of Kdm3a caused problems with quantitative PCR detection in the susceptible strain through reduced amplification efficiency due to altered secondary cDNA structure. Shape Probability Shift analysis predicted that the SNP often affects RNA folding; thus, it may impact protein translation. Despite these differences in rats, genetic knockout of Kdm3a in mice resulted in no dramatic effect on immune system development and activation or EAE susceptibility and severity. These results provide support for tools that analyze causative SNPs that impact nucleic acid structures. PMID:24312603

  4. Germ line transcription in mice bearing neor gene downstream of Igamma3 exon in the Ig heavy chain locus.

    PubMed

    Samara, Maha; Oruc, Zeliha; Dougier, Hei-Lanne; Essawi, Tamer; Cogné, Michel; Khamlichi, Ahmed Amine

    2006-04-01

    Class switch recombination (CSR) is preceded by germ line transcription that initiates from promoters upstream of switch (S) sequences and terminates downstream of associated constant genes. Previous work showed that germ line transcripts and their processing are required for CSR and that germ line transcription is regulated in a major part by a regulatory region located downstream of the Ig heavy chain locus. This long-range, polarized effect can be disturbed by inserting an expressed neomycine resistance (neo(r)) gene. To contribute to a better understanding of the mechanism of such a long-distance regulation, we generated knock-in mice in which a neo(r) gene was inserted downstream of Igamma3 exon leaving intact all the necessary elements for germ line transcription and splicing. We show that the expressed neo(r) gene interferes with transcription initiation from Igamma3, and that it impairs but does not block S recombination to Cgamma3. Moreover, we show for the first time that the neo(r) gene provides through chimeric neo(r)-Cgamma3 transcripts the necessary elements for splicing of germ line transcripts by activating two novel cryptic splice sites, one in the coding region of the intronless neo(r) gene and the other in the Igamma3-Cgamma3 intron.

  5. X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15.

    PubMed

    Demirci, F Yesim K; Rigatti, Brian W; Wen, Gaiping; Radak, Amy L; Mah, Tammy S; Baic, Corrine L; Traboulsi, Elias I; Alitalo, Tiina; Ramser, Juliane; Gorin, Michael B

    2002-04-01

    X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.

  6. An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs

    PubMed Central

    Fyfe, John C.; Hemker, Shelby L.; Venta, Patrick J.; Fitzgerald, Caitlin A.; Outerbridge, Catherine A.; Myers, Sherry L.; Giger, Urs

    2013-01-01

    Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9 Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome. PMID:23746554

  7. Analysis of mutations of MDR3 exons 9 and 23 in infants with parenteral nutrition-associated cholestasis

    PubMed Central

    YANG, XIU-FANG; LIU, GUO-SHENG; LI, MIN-XU

    2015-01-01

    The aim of this study was to investigate mutations of multidrug resistance 3 (MDR3) exons 9 and 23 in infants with parenteral nutrition-associated cholestasis (PNAC). A total of 41 infants with PNAC were enrolled in the study. Genomic DNA was extracted from the peripheral venous blood leukocytes of each patient and MDR3 exons 9 and 23 were amplified by polymerase chain reaction. One patient was identified who carried a frameshift mutation in MDR3 exon 23 (C.2793) that was caused by the insertion of a single adenine residue, while mutations were not found in MDR3 exon 23 in the other 40 patients. The clinical features of the patient with the MDR3 exon 23 frameshift mutation included high serum γ-glutamyl transferase levels, the absence of biliary dilatation and deformity in magnetic resonance cholangiopancreatography, and abnormal electrical capacitance tomography imaging of the liver. No mutations in MDR3 exon 9 were identified in any of the patients. All 41 PNAC patients recovered following oral ursodeoxycholic acid treatment. The C.2793 frameshift mutation in MDR3 exon 23 is potentially associated with the development of PNAC in infants. PMID:26668642

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

    PubMed

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

    2009-01-01

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

  9. Assessment of exonic single nucleotide polymorphisms in the adenosine A2A receptor gene to high myopia susceptibility in Chinese subjects

    PubMed Central

    Chen, Xiaoyan; Xue, Anquan; Chen, Wei; Ding, Yang; Yan, Dongsheng; Peng, Jiqing; Zeng, Changqing; Qu, Jia

    2011-01-01

    Purpose The adenosine A2A receptor (A2AR) modulates collagen synthesis and extracellular matrix production in ocular tissues that contribute to eye growth and the development of myopia. We aimed to determine if single nucleotide polymorphisms (SNPs) in A2AR exons associates with high myopia found in Chinese subjects. Methods DNA samples were prepared from venous lymphocytes of 175 Chinese subjects with high myopia of less than –8.00 diopters (D) correction and 101 ethnically similar controls with between –1.00 D and +1.00 D correction. The coding region sequences of A2AR were amplified by PCR and analyzed by Sanger sequencing. The detected variations were confirmed by reverse sequencing. Allelic frequencies of all detected common SNPs were assessed for Hardy–Weinberg equilibrium. Results Five variations in A2AR exons, 5675 A>G, 5765 C>T, 13325 G>A, 13448 C>T, and 14000 T>A, were detected in controls at a low frequency (<1%). However, one SNP, 13772 T>C (rs5751876), showed its polymorphism in 53.3% of the total study population. The rs5751876 is a synonymous substitution located in a tyrosine codon of exon 2. Despite no significant difference in genotype distribution between cases and controls, the frequency of heterozygotes with the rs5751876 genotype was significantly lower in subjects with high myopia. Conclusions The reduced frequency of the heterozygote rs5751876 genotype in subjects suggests a possible association of A2AR with high myopia in a Chinese population. PMID:22740769

  10. Detection of new HLA-DPB1 alleles generated by interallelic gene conversion using PCR amplification of DPB1 second exon sequences from sperm

    SciTech Connect

    Erlich, H.; Zangenberg, G.; Bugawan, T.

    1994-09-01

    The rate at which allelic diversity at the HLA class I and class II loci evolves has been the subject of considerable controversy as have the mechanisms which generate new alleles. The patchwork pattern of polymorphism, particularly within the second exon of the HLA-DPB1 locus where the polymorphic sequence motifs are localized to 6 discrete regions, is consistent with the hypothesis that much of the allelic sequence variation may have been generated by segmental exchange (gene conversion). To measure the rate of new DPB1 variant generation, we have developed a strategy in which DPB1 second exon sequences are amplified from pools of FACS-sorted sperm (n=50) from a heterozygous sperm donor. Pools of sperm from these heterozygous individuals are amplified with an allele-specific primer for one allele and analyzed with sequence-specific oligonucleotide probes (SSOP) complementary to the other allele. This screening procedure, which is capable of detecting a single variant molecule in a pool of parental alleles, allows the identification of new variants that have been generated by recombination and/or gene conversion between the two parental alleles. To control for potential PCR artifacts, the same screening procedure was carried out with mixtures of sperm from DPB1 *0301/*0301 and DPB1 *0401/ 0401 individuals. Pools containing putative new variants DPB1 alleles were analyzed further by cloning into M13 and sequencing the M13 clones. Our current estimate is that about 1/10,000 sperm from these heterozygous individuals represents a new DPB1 allele generated by micro-gene conversion within the second exon.

  11. ExSurv: A Web Resource for Prognostic Analyses of Exons Across Human Cancers Using Clinical Transcriptomes.

    PubMed

    Hashemikhabir, Seyedsasan; Budak, Gungor; Janga, Sarath Chandra

    2016-01-01

    Survival analysis in biomedical sciences is generally performed by correlating the levels of cellular components with patients' clinical features as a common practice in prognostic biomarker discovery. While the common and primary focus of such analysis in cancer genomics so far has been to identify the potential prognostic genes, alternative splicing - a posttranscriptional regulatory mechanism that affects the functional form of a protein due to inclusion or exclusion of individual exons giving rise to alternative protein products, has increasingly gained attention due to the prevalence of splicing aberrations in cancer transcriptomes. Hence, uncovering the potential prognostic exons can not only help in rationally designing exon-specific therapeutics but also increase specificity toward more personalized treatment options. To address this gap and to provide a platform for rational identification of prognostic exons from cancer transcriptomes, we developed ExSurv (https://exsurv.soic.iupui.edu), a web-based platform for predicting the survival contribution of all annotated exons in the human genome using RNA sequencing-based expression profiles for cancer samples from four cancer types available from The Cancer Genome Atlas. ExSurv enables users to search for a gene of interest and shows survival probabilities for all the exons associated with a gene and found to be significant at the chosen threshold. ExSurv also includes raw expression values across the cancer cohort as well as the survival plots for prognostic exons. Our analysis of the resulting prognostic exons across four cancer types revealed that most of the survival-associated exons are unique to a cancer type with few processes such as cell adhesion, carboxylic, fatty acid metabolism, and regulation of T-cell signaling common across cancer types, possibly suggesting significant differences in the posttranscriptional regulatory pathways contributing to prognosis. PMID:27528797

  12. ExSurv: A Web Resource for Prognostic Analyses of Exons Across Human Cancers Using Clinical Transcriptomes

    PubMed Central

    Hashemikhabir, Seyedsasan; Budak, Gungor; Janga, Sarath Chandra

    2016-01-01

    Survival analysis in biomedical sciences is generally performed by correlating the levels of cellular components with patients’ clinical features as a common practice in prognostic biomarker discovery. While the common and primary focus of such analysis in cancer genomics so far has been to identify the potential prognostic genes, alternative splicing – a posttranscriptional regulatory mechanism that affects the functional form of a protein due to inclusion or exclusion of individual exons giving rise to alternative protein products, has increasingly gained attention due to the prevalence of splicing aberrations in cancer transcriptomes. Hence, uncovering the potential prognostic exons can not only help in rationally designing exon-specific therapeutics but also increase specificity toward more personalized treatment options. To address this gap and to provide a platform for rational identification of prognostic exons from cancer transcriptomes, we developed ExSurv (https://exsurv.soic.iupui.edu), a web-based platform for predicting the survival contribution of all annotated exons in the human genome using RNA sequencing-based expression profiles for cancer samples from four cancer types available from The Cancer Genome Atlas. ExSurv enables users to search for a gene of interest and shows survival probabilities for all the exons associated with a gene and found to be significant at the chosen threshold. ExSurv also includes raw expression values across the cancer cohort as well as the survival plots for prognostic exons. Our analysis of the resulting prognostic exons across four cancer types revealed that most of the survival-associated exons are unique to a cancer type with few processes such as cell adhesion, carboxylic, fatty acid metabolism, and regulation of T-cell signaling common across cancer types, possibly suggesting significant differences in the posttranscriptional regulatory pathways contributing to prognosis. PMID:27528797

  13. Mice with an isoform-ablating Mecp2 exon 1 mutation recapitulate the neurologic deficits of Rett syndrome.

    PubMed

    Yasui, Dag H; Gonzales, Michael L; Aflatooni, Justin O; Crary, Florence K; Hu, Daniel J; Gavino, Bryant J; Golub, Mari S; Vincent, John B; Carolyn Schanen, N; Olson, Carl O; Rastegar, Mojgan; Lasalle, Janine M

    2014-05-01

    Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT OMIM 312750). Alternative inclusion of MECP2/Mecp2 exon 1 with exons 3 and 4 encodes MeCP2-e1 or MeCP2-e2 protein isoforms with unique amino termini. While most MECP2 mutations are located in exons 3 and 4 thus affecting both isoforms, MECP2 exon 1 mutations but not exon 2 mutations have been identified in RTT patients, suggesting that MeCP2-e1 deficiency is sufficient to cause RTT. As expected, genetic deletion of Mecp2 exons 3 and/or 4 recapitulates RTT-like neurologic defects in mice. However, Mecp2 exon 2 knockout mice have normal neurologic function. Here, a naturally occurring MECP2 exon 1 mutation is recapitulated in a mouse model by genetic engineering. A point mutation in the translational start codon of Mecp2 exon 1, transmitted through the germline, ablates MeCP2-e1 translation while preserving MeCP2-e2 production in mouse brain. The resulting MeCP2-e1 deficient mice developed forelimb stereotypy, hindlimb clasping, excessive grooming and hypo-activity prior to death between 7 and 31 weeks. MeCP2-e1 deficient mice also exhibited abnormal anxiety, sociability and ambulation. Despite MeCP2-e1 and MeCP2-e2 sharing, 96% amino acid identity, differences were identified. A fraction of phosphorylated MeCP2-e1 differed from the bulk of MeCP2 in subnuclear localization and co-factor interaction. Furthermore, MeCP2-e1 exhibited enhanced stability compared with MeCP2-e2 in neurons. Therefore, MeCP2-e1 deficient mice implicate MeCP2-e1 as the sole contributor to RTT with non-redundant functions.

  14. POLYMORPHISM IN THE CODING REGION SEQUENCE OF GDF8 GENE IN INDIAN SHEEP.

    PubMed

    Pothuraju, M; Mishra, S K; Kumar, S N; Mohamed, N F; Kataria, R S; Yadav, D K; Arora, R

    2015-11-01

    The present study was undertaken to identify polymorphism in the coding sequence of GDF8gene across indigenous meat type sheep breeds. A 1647 bp sequence was generated, encompassing 208 bp of the 5'UTR, 1128 bp of coding region (exon1, 2 and 3) as well as 311 bp of 3'UTR. The sheep and goat GDF8 gene sequences were observed to be highly conserved as compared to cattle, buffalo, horse and pig. Several nucleotide variations were observed across coding sequence of GDF8 gene in Indian sheep. Three polymorphic sites were identified in the 5'UTR, one in exon 1 and one in the exon 2 regions. Both SNPs in the exonic region were found to be non-synonymous. The mutations c.539T > G and c.821T > A discovered in this study in the exon 1 and exon 2, respectively, have not been previously reported. The information generated provides preliminary indication of the functional diversity present in Indian sheep at the coding region of GDF8gene. The novel as well as the previously reported SNPs discovered in the Indian sheep warrant further analysis to see whether they affect the phenotype. Future studies will need to establish the affect of reported SNPs in the expression of the GDF8 gene in Indian sheep population. PMID:26845859

  15. Characterization of microsatellites in the coding regions

    SciTech Connect

    Tuskan, Gerald A; Li, Shuxian; Yin, Tongming; Wang, Prof. Mingxiu

    2009-01-01

    With the development of high-throughput sequencing techniques, transcriptome sequencing projects which provide valuable resources for designing simple sequence repeat (SSR) primers have been carried out for many plants. However, the utility of SSRs for molecular breeding depends on genomewide distribution and coverage, as well as moderately high allelic variability, in the available SSR library. In this study, we characterized the exonic SSRs developed from the publicly available Populus genome as a case study to determine their value for molecular breeding. As expected, our results confirmed that microsatellites occurred approximately three times less often in coding regions than in non-coding regions. Mutability test also showed that exonic SSRs contained less allelic variability than intronic SSRs. More importantly, exonic SSRs were unevenly distributed both among and within chromosomes. Large exonic SSRs deserts were observed on several chromosomes. Differential selection between paralogous chromosomes, at the gene level, appears to be responsible for these SSR deserts, though the mechanisms that cause chromosome-specific SSR deserts are not known. This work provides ample evidence that the candidate gene approach based on unigenes identified from transcribed sequences may not be the best strategy to identify highly polymorphic SSRs.

  16. TP53 exon-6 truncating mutations produce separation of function isoforms with pro-tumorigenic functions

    PubMed Central

    Shirole, Nitin H; Pal, Debjani; Kastenhuber, Edward R; Senturk, Serif; Boroda, Joseph; Pisterzi, Paola; Miller, Madison; Munoz, Gustavo; Anderluh, Marko; Ladanyi, Marc; Lowe, Scott W; Sordella, Raffaella

    2016-01-01

    TP53 truncating mutations are common in human tumors and are thought to give rise to p53-null alleles. Here, we show that TP53 exon-6 truncating mutations occur at higher than expected frequencies and produce proteins that lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis. Functionally and molecularly, these p53 mutants resemble the naturally occurring alternative p53 splice variant, p53-psi. Accordingly, these mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). Together, our studies reveal that TP53 exon-6 truncating mutations, contrary to current beliefs, act beyond p53 loss to promote tumorigenesis, and could inform the development of strategies to target cancers driven by these prevalent mutations. DOI: http://dx.doi.org/10.7554/eLife.17929.001 PMID:27759562

  17. The mouse formin (Fmn) gene: Genomic structure, novel exons, and genetic mapping

    SciTech Connect

    Wang, C.C.; Chan, D.C.; Leder, P.

    1997-02-01

    Mutations in the mouse formin (Fmn) gene, formerly known as the limb deformity (ld) gene, give rise to recessively inherited limb deformities and renal malformations or aplasia. The Fmn gene encodes many differentially processed transcripts that are expressed in both adult and embryonic tissues. To study the genomic organization of the Fmn locus, we have used Fmn probes to isolate and characterize genomic clones spanning 500 kb. Our analysis of these clones shows that the Fmn gene is composed of at least 24 exons and spans 400 kb. We have identified two novel exons that are expressed in the developing embryonic limb bud as well as adult tissues such as brain and kidney. We have also used a microsatellite polymorphism from within the Fmn gene to map it genetically to a 2.2-cM interval between D2Mit58 and D2Mit103. 36 refs., 6 figs., 1 tab.

  18. Variable intron/exon structure in the oligochaete lombricine kinase gene.

    PubMed

    Doumen, Chris

    2012-09-01

    Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. PMID:22705027

  19. Large exonic deletions in POLR3B gene cause POLR3-related leukodystrophy.

    PubMed

    Gutierrez, Mariana; Thiffault, Isabelle; Guerrero, Kether; Martos-Moreno, Gabriel Á; Tran, Luan T; Benko, William; van der Knaap, Marjo S; van Spaendonk, Rosalina M L; Wolf, Nicole I; Bernard, Geneviève

    2015-06-05

    POLR3-related (or 4H) leukodystrophy is an autosomal recessive disorder caused by mutations in POLR3A or POLR3B and is characterized by neurological and non-neurological features. In a small proportion of patients, no mutation in either gene or only one mutation is found. Analysis of the POLR3B cDNA revealed a large deletion of exons 21-22 in one case and of exons 26-27 in another case. These are the first reports of long deletions causing POLR3-related leukodystrophy, suggesting that deletions and duplications in POLR3A or POLR3B should be investigated in patients with a compatible phenotype, especially if one pathogenic variant has been identified.

  20. Two novel mutations on exon 8 and intron 65 of COL7A1 gene in two Chinese brothers result in recessive dystrophic epidermolysis bullosa.

    PubMed

    Lin, Ying; Chen, Xue-Jun; Liu, Wei; Gong, Bo; Xie, Jun; Xiong, Jun-Hao; Cheng, Jing; Duan, Xi-Ling; Lin, Zhao-Chun; Huang, Lu-Lin; Wan, Hui-Ying; Liu, Xiao-Qi; Song, Lin-Hong; Yang, Zheng-Lin

    2012-01-01

    Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen - the main component of the anchoring fibrils at the dermal-epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5' donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control

  1. A novel exon 3 mutation in a Tunisian patient with Lafora's disease.

    PubMed

    Khiari, H Mrabet; Lesca, G; Malafosse, A; Mrabet, A

    2011-05-15

    We report a Tunisian patient born from consanguineous marriage affected with progressive myoclonus epilepsy and cognitive decline, consistent with the diagnosis of Lafora disease. Genetic analysis showed a novel c.659 T>A mutation on exon 3 of the EPM2A gene, converting a leucine to a glutamine residue at amino acid position 220 (p.Leu220Gln), in the dual-specificity phosphatase domain. PMID:21371719

  2. Selective Blockade of Periostin Exon 17 Preserves Cardiac Performance in Acute Myocardial Infarction.

    PubMed

    Taniyama, Yoshiaki; Katsuragi, Naruto; Sanada, Fumihiro; Azuma, Junya; Iekushi, Kazuma; Koibuchi, Nobutaka; Okayama, Keita; Ikeda-Iwabu, Yuka; Muratsu, Jun; Otsu, Rei; Rakugi, Hiromi; Morishita, Ryuichi

    2016-02-01

    We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon 17) and Pn-4 (lacking exons 17 and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon 17 in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming growth factor-β1. Importantly, the number of α-smooth muscle actin-positive myofibroblasts was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited fibroblast and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-17, via a neutralizing antibody or drug, without suppressing other periostin variants might offer a new class of medication for the treatment of acute myocardial infarction patients.

  3. DVL1 frameshift mutations clustering in the penultimate exon cause autosomal-dominant Robinow syndrome.

    PubMed

    White, Janson; Mazzeu, Juliana F; Hoischen, Alexander; Jhangiani, Shalini N; Gambin, Tomasz; Alcino, Michele Calijorne; Penney, Samantha; Saraiva, Jorge M; Hove, Hanne; Skovby, Flemming; Kayserili, Hülya; Estrella, Elicia; Vulto-van Silfhout, Anneke T; Steehouwer, Marloes; Muzny, Donna M; Sutton, V Reid; Gibbs, Richard A; Lupski, James R; Brunner, Han G; van Bon, Bregje W M; Carvalho, Claudia M B

    2015-04-01

    Robinow syndrome is a genetically heterogeneous disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features and for which both autosomal-recessive and autosomal-dominant inheritance patterns have been described. Causative variants in the non-canonical signaling gene WNT5A underlie a subset of autosomal-dominant Robinow syndrome (DRS) cases, but most individuals with DRS remain without a molecular diagnosis. We performed whole-exome sequencing in four unrelated DRS-affected individuals without coding mutations in WNT5A and found heterozygous DVL1 exon 14 mutations in three of them. Targeted Sanger sequencing in additional subjects with DRS uncovered DVL1 exon 14 mutations in five individuals, including a pair of monozygotic twins. In total, six distinct frameshift mutations were found in eight subjects, and all were heterozygous truncating variants within the penultimate exon of DVL1. In five families in which samples from unaffected parents were available, the variants were demonstrated to represent de novo mutations. All variant alleles are predicted to result in a premature termination codon within the last exon, escape nonsense-mediated decay (NMD), and most likely generate a C-terminally truncated protein with a distinct -1 reading-frame terminus. Study of the transcripts extracted from affected subjects' leukocytes confirmed expression of both wild-type and variant alleles, supporting the hypothesis that mutant mRNA escapes NMD. Genomic variants identified in our study suggest that truncation of the C-terminal domain of DVL1, a protein hypothesized to have a downstream role in the Wnt-5a non-canonical pathway, is a common cause of DRS. PMID:25817016

  4. A homozygous deletion of exon 1 in WISP3 causes progressive pseudorheumatoid dysplasia in two siblings

    PubMed Central

    Neerinckx, Barbara; Thues, Cedric; Wouters, Carine; Lechner, Sarah; Westhovens, Rene; Van Esch, Hilde

    2015-01-01

    Progressive pseudorheumatoid dysplasia (PPD) is a rare autosomal recessive disease that causes progressive joint stiffness and pain. It is associated with loss-of-function mutations in the WISP3 gene. We describe two sisters suffering from PPD in whom molecular genetic analysis revealed a homozygous deletion of exon 1 and of the 5′UTR of the WISP3 gene. This is the first time that a gross deletion has been described as the causal mutation in PPD. PMID:27081554

  5. Intracellular Folding of the Tetrahymena Group I Intron Depends on Exon Sequence and Promoter Choice

    SciTech Connect

    Koduvayur,S.; Woodson, S.

    2004-01-01

    The Tetrahymena group I intron splices 20 to 50 times faster in Tetrahymena than in vitro, implying that the intron rapidly adopts its active conformation in the cell. The importance of cotranscriptional folding and the contribution of the rRNA exons to the stability of the active pre-RNA structure were investigated by comparing the activity of minimal pre-RNAs expressed in Escherichia coli. Pre-RNAs containing exons derived from E. coli 23 S rRNA were three to four times more active than the wild-type Tetrahymena pre-RNA. E. coli transcripts of the chimeric E. coli pre-RNA were two to eight times more active than were T7 transcripts. However, the effect of cotranscriptional folding depends on exon sequences. Unexpectedly, the unspliced pre-RNA decays more slowly than predicted from the rate of splicing. This observation is best explained by partitioning of transcripts into active and inactive pools. We propose that the active pool splices within a few seconds, whereas the inactive pool is degraded without appreciable splicing.

  6. A New Exon Derived from a Mammalian Apparent LTR Retrotransposon of the SUPT16H Gene

    PubMed Central

    Bae, Min-In; Kim, Yun-Ji; Lee, Ja-Rang; Jung, Yi-Deun; Kim, Heui-Soo

    2013-01-01

    The SUPT16H gene known as FACTP140 is required for the transcription of other genes. For transcription, genes need to be complexed with accessory factors, including transcription factors and RNA polymerase II. One such factor, FACT, interacts with histones H2A/H2B for nucleosome disassembly and transcription elongation. The SUPT16H gene has a transcript and many expressed sequence tags (ESTs). We were especially interested in an MaLR-derived transcript (EST, BX333035) that included a new exon introduced by a transposable element, a mammalian apparent LTR retrotransposon (MaLR). The MaLR was detected ranging from humans to galagos, indicating the MaLR in the SUPT16H gene is integrated into the primate ancestor genome. A new exon was created by alternative donor site provided by the MaLR. The original transcript and the MaLR-derived transcript were expressed in various human, rhesus monkey, and other primate tissues. Additionally, we identified a new alternative transcript that included the MaLR, but there was no significant difference in the expression of the original transcript and the MaLR-derived transcript. Interestingly, the new alternative transcript and the MaLR-derived transcript had the MaLR sequence in the new exon, but they had different structures by adopting different 3′ splice sites. From this study, we verified transposable elements that contributed to transcriptome diversity. PMID:23671841

  7. Chemical and mechanistic toxicology evaluation of exon skipping phosphorodiamidate morpholino oligomers in mdx mice.

    PubMed

    Sazani, Peter; Ness, Kirk P Van; Weller, Doreen L; Poage, Duane; Nelson, Keith; Shrewsbury, And Stephen B

    2011-05-01

    AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) designed to induce skipping of dystrophin exon 51 and restore its expression in patients with Duchenne muscular dystrophy (DMD). Preclinically, restoration of dystrophin in the dystrophic mdx mouse model requires skipping of exon 23, achieved with the mouse-specific PMO, AVI-4225. Herein, we report the potential toxicological consequences of exon skipping and dystrophin restoration in mdx mice using AVI-4225. We also evaluated the toxicological effects of AVI-4658 in both mdx and wild-type mice. In both studies, animals were dosed once weekly for 12 weeks up to the maximum feasible dose of 960 mg/kg per injection. Both AVI-4658 and AVI-4225 were well-tolerated at all doses. Findings in AVI-4225-treated animals were generally limited to mild renal tubular basophilia/vacuolation, without any significant changes in renal function and with evidence of reversing. No toxicity associated with the mechanism of action of AVI-4225 in a dystrophic animal was observed.

  8. How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing?

    PubMed

    Hollander, Dror; Naftelberg, Shiran; Lev-Maor, Galit; Kornblihtt, Alberto R; Ast, Gil

    2016-10-01

    The splice sites (SSs) delimiting an intron are brought together in the earliest step of spliceosome assembly yet it remains obscure how SS pairing occurs, especially when introns are thousands of nucleotides long. Splicing occurs in vivo in mammals within minutes regardless of intron length, implying that SS pairing can instantly follow transcription. Also, factors required for SS pairing, such as the U1 small nuclear ribonucleoprotein (snRNP) and U2AF65, associate with RNA polymerase II (RNAPII), while nucleosomes preferentially bind exonic sequences and associate with U2 snRNP. Based on recent publications, we assume that the 5' SS-bound U1 snRNP can remain tethered to RNAPII until complete synthesis of the downstream intron and exon. An additional U1 snRNP then binds the downstream 5' SS, whereas the RNAPII-associated U2AF65 binds the upstream 3' SS to facilitate SS pairing along with exon definition. Next, the nucleosome-associated U2 snRNP binds the branch site to advance splicing complex assembly. This may explain how RNAPII and chromatin are involved in spliceosome assembly and how introns lengthened during evolution with a relatively minimal compromise in splicing.

  9. Pelizaeus-Merzbacher disease: tight linkage to proteolipid protein gene exon variant.

    PubMed Central

    Trofatter, J A; Dlouhy, S R; DeMyer, W; Conneally, P M; Hodes, M E

    1989-01-01

    Pelizaeus-Merzbacher disease (PMD) is a human X chromosome-linked dysmyelination disorder of the central nervous system for which the genetic defect has not yet been established. The jimpy mutation jp of the mouse is an X chromosome-linked disorder of myelin formation. The mutation is at an intron/exon splice site in the mouse gene for proteolipid protein (PLP). With the jimpy mouse mutation as a precedent, we focused our attention on the human PLP gene, which is found at Xq22. The polymerase chain reaction was used to amplify the exons of the PLP gene of an affected male from a large Indiana PMD kindred. DNA sequencing showed a C----T transition at nucleotide 40 of the second exon. An affected third cousin also showed this sequence variation, while two unaffected male relatives (sons of an obligate carrier female) had the normal cytidine nucleotide. Allele-specific oligonucleotides were used to generate data for linkage studies on the above mentioned PMD kindred. Our results show tight linkage (theta = 0) of PMD to PLP with a lod (logarithm of odds) score of 4.62. In six other unrelated PMD kindreds, only the normal-sequence oligonucleotide hybridized, which indicates genetic heterogeneity. The radical nature of the predicted amino acid change (proline to leucine), suggests that the PMD-causing defect may have been delineated in one kindred. Images PMID:2480601

  10. Evolutionary analysis of the mammalian M1 aminopeptidases reveals conserved exon structure and gene death.

    PubMed

    Maynard, Karen Beasley; Smith, Shannon A; Davis, Anthony C; Trivette, Andrew; Seipelt-Thiemann, Rebecca L

    2014-11-15

    The members of the M1 aminopeptidase family share conserved domains, yet show functional divergence within the family as a whole. In order to better understand this family, this study analyzed the mammalian members in depth at exon, gene, and protein levels. The twelve human members, eleven rat members, and eleven mouse members were first analyzed in multiple alignments to visualize both reported and unreported conserved domains. Phylogenetic trees were then generated for humans, rats, mice, and all mammals to determine how closely related the homologs were and to gain insight to the divergence in the family members. This produced three groups with similarity within the family. Next, a synteny study was completed to determine the present locations of the genes and changes that had occurred. It became apparent that gene death likely resulted in the lack of one member in mouse and rat. Finally, an in-depth analysis of the exon structure revealed that nine members of the human family and eight in mouse, are highly conserved within the exon structure. Taken together, these results indicate that the M1 aminopeptidase family is a divergent family with three subgroups and that genetic evidence mirrors categorization of the family by enzymatic function.

  11. MED12 exon 2 mutations in phyllodes tumors of the breast.

    PubMed

    Nagasawa, Satoi; Maeda, Ichiro; Fukuda, Takayo; Wu, Wenwen; Hayami, Ryosuke; Kojima, Yasuyuki; Tsugawa, Ko-Ichiro; Ohta, Tomohiko

    2015-07-01

    Exon 2 of MED12, a subunit of the transcriptional mediator complex, has been frequently mutated in uterine leiomyomas and breast fibroadenomas; however, it has been rarely mutated in other tumors. Although the mutations were also found in uterine leiomyosarcomas, the frequency was significantly lower than in uterine leiomyomas. Here, we examined the MED12 mutation in phyllodes tumors, another biphasic tumor with epithelial and stromal components related to breast fibroadenomas. Mutations in MED12 exon 2 were analyzed in nine fibroadenomas and eleven phyllodes tumors via Sanger sequencing. A panel of cancer- and sarcoma-related genes was also analyzed using Ion Torrent next-generation sequencing. Six mutations in fibroadenomas, including those previously reported (6/9, 67%), and five mutations in phyllodes tumors (5/11, 45%) were observed. Three mutations in the phyllodes tumors were missense mutations at Gly44, which is common in uterine leiomyomas and breast fibroadenomas. In addition, two deletion mutations (in-frame c.133_144del12 and loss of splice acceptor c.100-68_137del106) were observed in the phyllodes tumors. No other recurrent mutation was observed with next-generation sequencing. Frequent mutations in MED12 exon 2 in the phyllodes tumors suggest that it may share genetic etiology with uterine leiomyoma, a subgroup of uterine leiomyosarcomas and breast fibroadenoma.

  12. Subfunctionalization of duplicate mitf genes associated with differential degeneration of alternative exons in fish.

    PubMed Central

    Altschmied, Joachim; Delfgaauw, Jacqueline; Wilde, Brigitta; Duschl, Jutta; Bouneau, Laurence; Volff, Jean-Nicolas; Schartl, Manfred

    2002-01-01

    The microphthalmia-associated transcription factor (MITF) exists in at least four isoforms. These are generated in higher vertebrates using alternative 5' exons and promoters from a single gene. Two separate genes (mitf-m and mitf-b), however, are present in different teleost fish species including the poeciliid Xiphophorus, the pufferfishes Fugu rubripes and Tetraodon nigroviridis, and the zebrafish Danio rerio. Fish proteins MITF-m and MITF-b correspond at both the structural and the expression levels to one particular bird/mammalian MITF isoform. In the teleost lineage subfunctionalization of mitf genes after duplication at least 100 million years ago is associated with the degeneration of alternative exons and, probably, regulatory elements and promoters. For example, a remnant of the first exon specific for MITF-m is detected within the pufferfish gene encoding MITF-b. Retracing the evolutionary history of mitf genes in vertebrates uncovered the differential recruitment of new introns specific for either the teleost or the bird/mammalian lineage. PMID:12019239

  13. The exon junction complex is required for definition and excision of neighboring introns in Drosophila.

    PubMed

    Hayashi, Rippei; Handler, Dominik; Ish-Horowicz, David; Brennecke, Julius

    2014-08-15

    Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events. PMID:25081352

  14. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  15. Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy

    PubMed Central

    Gramlich, Michael; Pane, Luna Simona; Zhou, Qifeng; Chen, Zhifen; Murgia, Marta; Schötterl, Sonja; Goedel, Alexander; Metzger, Katja; Brade, Thomas; Parrotta, Elvira; Schaller, Martin; Gerull, Brenda; Thierfelder, Ludwig; Aartsma-Rus, Annemieke; Labeit, Siegfried; Atherton, John J; McGaughran, Julie; Harvey, Richard P; Sinnecker, Daniel; Mann, Matthias; Laugwitz, Karl-Ludwig; Gawaz, Meinrad Paul; Moretti, Alessandra

    2015-01-01

    Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM. PMID:25759365

  16. Plug-and-Play Genetic Access to Drosophila Cell Types Using Exchangeable Exon Cassettes

    PubMed Central

    Diao, Fengqiu; Ironfield, Holly; Luan, Haojiang; Diao, Feici; Shropshire, William C.; Ewer, John; Marr, Elizabeth; Potter, Christopher J.; Landgraf, Matthias; White, Benjamin H.

    2015-01-01

    Summary Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here we introduce a simple, versatile method for achieving cell type-specific expression of transgenes that leverages the untapped potential of “coding introns” (i.e. introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted “plug-and-play” cassettes (called “Trojan exons”) that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system. PMID:25732830

  17. Dynamic ASXL1 Exon Skipping and Alternative Circular Splicing in Single Human Cells

    PubMed Central

    Natarajan, Sivaraman; Carter, Robert; Brown, Patrick O.

    2016-01-01

    Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology. PMID:27736885

  18. How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing?

    PubMed

    Hollander, Dror; Naftelberg, Shiran; Lev-Maor, Galit; Kornblihtt, Alberto R; Ast, Gil

    2016-10-01

    The splice sites (SSs) delimiting an intron are brought together in the earliest step of spliceosome assembly yet it remains obscure how SS pairing occurs, especially when introns are thousands of nucleotides long. Splicing occurs in vivo in mammals within minutes regardless of intron length, implying that SS pairing can instantly follow transcription. Also, factors required for SS pairing, such as the U1 small nuclear ribonucleoprotein (snRNP) and U2AF65, associate with RNA polymerase II (RNAPII), while nucleosomes preferentially bind exonic sequences and associate with U2 snRNP. Based on recent publications, we assume that the 5' SS-bound U1 snRNP can remain tethered to RNAPII until complete synthesis of the downstream intron and exon. An additional U1 snRNP then binds the downstream 5' SS, whereas the RNAPII-associated U2AF65 binds the upstream 3' SS to facilitate SS pairing along with exon definition. Next, the nucleosome-associated U2 snRNP binds the branch site to advance splicing complex assembly. This may explain how RNAPII and chromatin are involved in spliceosome assembly and how introns lengthened during evolution with a relatively minimal compromise in splicing. PMID:27507607

  19. The Status of Exon Skipping as a Therapeutic Approach to Duchenne Muscular Dystrophy

    PubMed Central

    Lu, Qi-Long; Yokota, Toshifumi; Takeda, Shin'ichi; Garcia, Luis; Muntoni, Francesco; Partridge, Terence

    2011-01-01

    Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level. PMID:20978473

  20. Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

    PubMed Central

    Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

    2012-01-01

    Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

  1. First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome.

    PubMed

    Beygo, J; Buiting, K; Seland, S; Lüdecke, H-J; Hehr, U; Lich, C; Prager, B; Lohmann, D R; Wieczorek, D

    2012-01-01

    Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1.

  2. NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis

    PubMed Central

    Dolata, Jakub; Guo, Yanwu; Kołowerzo, Agnieszka; Smoliński, Dariusz; Brzyżek, Grzegorz; Jarmołowski, Artur; Świeżewski, Szymon

    2015-01-01

    The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5′ and 3′ splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1-dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis. PMID:25568310

  3. Two-Exon Skipping within MLPH Is Associated with Coat Color Dilution in Rabbits

    PubMed Central

    Lehner, Stefanie; Gähle, Marion; Dierks, Claudia; Stelter, Ricarda; Gerber, Jonathan; Brehm, Ralph; Distl, Ottmar

    2013-01-01

    Coat color dilution turns black coat color to blue and red color to cream and is a characteristic in many mammalian species. Matings among Netherland Dwarf, Loh, and Lionhead Dwarf rabbits over two generations gave evidence for a monogenic autosomal recessive inheritance of coat colour dilution. Histological analyses showed non-uniformly distributed, large, agglomerating melanin granules in the hair bulbs of coat color diluted rabbits. We sequenced the cDNA of MLPH in two dilute and one black rabbit for polymorphism detection. In both color diluted rabbits, skipping of exons 3 and 4 was present resulting in altered amino acids at p.QGL[37-39]QWA and a premature stop codon at p.K40*. Sequencing of genomic DNA revealed a c.111-5C>A splice acceptor mutation within the polypyrimidine tract of intron 2 within MLPH. This mutation presumably causes skipping of exons 3 and 4. In 14/15 dilute rabbits, the c.111-5C>A mutation was homozygous and in a further dilute rabbit, heterozygous and in combination with a homozygous frame shift mutation within exon 6 (c.585delG). In conclusion, our results demonstrated a colour dilution associated MLPH splice variant causing a strongly truncated protein (p.Q37QfsX4). An involvement of further MLPH-associated mutations needs further investigations. PMID:24376820

  4. Detection of clinically relevant exonic copy-number changes by array CGH.

    PubMed

    Boone, Philip M; Bacino, Carlos A; Shaw, Chad A; Eng, Patricia A; Hixson, Patricia M; Pursley, Amber N; Kang, Sung-Hae L; Yang, Yaping; Wiszniewska, Joanna; Nowakowska, Beata A; del Gaudio, Daniela; Xia, Zhilian; Simpson-Patel, Gayle; Immken, LaDonna L; Gibson, James B; Tsai, Anne C-H; Bowers, Jennifer A; Reimschisel, Tyler E; Schaaf, Christian P; Potocki, Lorraine; Scaglia, Fernando; Gambin, Tomasz; Sykulski, Maciej; Bartnik, Magdalena; Derwinska, Katarzyna; Wisniowiecka-Kowalnik, Barbara; Lalani, Seema R; Probst, Frank J; Bi, Weimin; Beaudet, Arthur L; Patel, Ankita; Lupski, James R; Cheung, Sau Wai; Stankiewicz, Pawel

    2010-12-01

    Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.

  5. Detection of Clinically Relevant Exonic Copy-Number Changes by Array CGH

    PubMed Central

    Boone, Philip M.; Bacino, Carlos A.; Shaw, Chad A.; Eng, Patricia A.; Hixson, Patricia M.; Pursley, Amber N.; Kang, Sung-Hae L.; Yang, Yaping; Wiszniewska, Joanna; Nowakowska, Beata A.; Gaudio, Daniela del; Xia, Zhilian; Simpson-Patel, Gayle; Immken, LaDonna L.; Gibson, James B.; Tsai, Anne C.-H.; Bowers, Jennifer A.; Reimschisel, Tyler E.; Schaaf, Christian P.; Potocki, Lorraine; Scaglia, Fernando; Gambin, Tomasz; Sykulski, Maciej; Bartnik, Magdalena; Derwinska, Katarzyna; Wisniowiecka-Kowalnik, Barbara; Lalani, Seema R.; Probst, Frank J.; Bi, Weimin; Beaudet, Arthur L.; Patel, Ankita; Lupski, James R.; Cheung, Sau Wai; Stankiewicz, Pawel

    2011-01-01

    Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes. PMID:20848651

  6. The exon junction complex is required for definition and excision of neighboring introns in Drosophila.

    PubMed

    Hayashi, Rippei; Handler, Dominik; Ish-Horowicz, David; Brennecke, Julius

    2014-08-15

    Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events.

  7. Differences in methylation patterns in the methylation boundary region of IDS gene in Hunter syndrome patients: implications for CpG hot spot mutations.

    PubMed

    Tomatsu, Shunji; Sukegawa, Kazuko; Trandafirescu, Georgeta G; Gutierrez, Monica A; Nishioka, Tatsuo; Yamaguchi, Seiji; Orii, Tadao; Froissart, Roseline; Maire, Irene; Chabas, Amparo; Cooper, Alan; Di Natale, Paola; Gal, Andreas; Noguchi, Akihiko; Sly, William S

    2006-07-01

    Hunter syndrome, an X-linked disorder, results from deficiency of iduronate-2-sulfatase (IDS). Around 40% of independent point mutations at IDS were found at CpG sites as transitional events. The 15 CpG sites in the coding sequences of exons 1 and 2, which are normally hypomethylated, account for very few of transitional mutations. By contrast, the CpG sites in the coding sequences of exon 3, though also normally hypomethylated, account for much higher fraction of transitional mutations. To better understand relationship between methylation status and CpG transitional mutations in this region, the methylation patterns of 11 Hunter patients with transitional mutations at CpG sites were investigated using bisulfite genomic sequencing. The patient cohort mutation spectrum is composed of one mutation in exon 1 (one patient) and three different mutations in exon 3 (10 patients). We confirmed that in normal males, cytosines at the CpG sites from the promoter region to a portion of intron 3 were hypomethylated. However, specific CpG sites in this area were more highly methylated in patients. The patients with p.R8X (exon 1), p.P86L (exon 3), and p.R88H (exon 3) mutations had a hypermethylated condition in exon 2 to intron 3 but retained hypomethylation in exon 1. The same trend was found in four patients with p.A85T (exon 3), although the degree of hypermethylation was less. These findings suggest methylation patterns in the beginning of IDS genomic region are polymorphic in humans and that hypermethylation in this region in some individuals predisposes them to CpG mutations resulting in Hunter syndrome.

  8. Molecular structure of the chicken vitamin D-induced calbindin-D28K gene reveals eleven exons, six Ca2+-binding domains, and numerous promoter regulatory elements.

    PubMed

    Minghetti, P P; Cancela, L; Fujisawa, Y; Theofan, G; Norman, A W

    1988-04-01

    The seco-steroid hormone 1,25-dihydroxyvitamin D3 is known to induce the expression of a calcium binding protein termed calbindin-D28K in a variety of target tissues. In order to comprehend the mechanism of induction we have cloned and sequenced the chicken calbindin-D28K gene. The gene spans some 18.5 kilobases (kb) of chromosomal DNA from the putative Cap site to the polyadenylation site of the 2.8 kb mRNA. It is split into 11 coding exons by 10 intervening sequences. The promoter region of this gene is markedly G + C-rich (60-80%) extending from -225 to +400. Within this region we find 70 CpG dinucleotides, four G-C boxes, and numerous known promoter regulatory signals. These putative regulatory signals include a TATA box (ATAAATA) at -30 and a CAT box (CCAAT) at -326. Ten additional variant CAT boxes are found in the upstream promoter region (-218 to -770) of this gene. Furthermore we have identified a glucocorticoid-like responsive element at -410 (TCTACACACTGTTCC) and this element overlaps a metal responsive element (TGCACTC) and a variant CAT box (CCAAAT) and juxtaposes an enhancer-like core element (AAATGGT) on its 3'-side. In addition, the calbindin-D28K promoter is composed of a variety of simple repeated sequences, some of which are components of putative regulatory signals. All splice junctions were found to conform to the GT-AG rule. A consensus sequence of the 5'-splice junction reads AG/GTAAG-TTATA. A consensus sequence of the 3'-splice site consists of two elements: a pyrimidine track (mainly T) followed by ACAG/G-T. A two-dimensional model of calbindin-D28K was constructed which projects the existence of 6 alpha-helix-loop-alpha-helix regions characteristic of calcium binding domains. The 3'-end of the gene consists of a single large (2039 base pair) uninterrupted exon, an organizational feature common to other members of the calcium binding protein gene family which include calmodulin, parvalbumin, Spec I, myosin light chains, etc. Another feature

  9. Association between the dopamine D4 receptor gene exon III variable number of tandem repeats and political attitudes in female Han Chinese.

    PubMed

    Ebstein, Richard P; Monakhov, Mikhail V; Lu, Yunfeng; Jiang, Yushi; Lai, Poh San; Chew, Soo Hong

    2015-08-22

    Twin and family studies suggest that political attitudes are partially determined by an individual's genotype. The dopamine D4 receptor gene (DRD4) exon III repeat region that has been extensively studied in connection with human behaviour, is a plausible candidate to contribute to individual differences in political attitudes. A first United States study provisionally identified this gene with political attitude along a liberal-conservative axis albeit contingent upon number of friends. In a large sample of 1771 Han Chinese university students in Singapore, we observed a significant main effect of association between the DRD4 exon III variable number of tandem repeats and political attitude. Subjects with two copies of the 4-repeat allele (4R/4R) were significantly more conservative. Our results provided evidence for a role of the DRD4 gene variants in contributing to individual differences in political attitude particularly in females and more generally suggested that associations between individual genes, and neurochemical pathways, contributing to traits relevant to the social sciences can be provisionally identified. PMID:26246555

  10. Association between the dopamine D4 receptor gene exon III variable number of tandem repeats and political attitudes in female Han Chinese

    PubMed Central

    Ebstein, Richard P.; Monakhov, Mikhail V.; Lu, Yunfeng; Jiang, Yushi; Lai, Poh San; Chew, Soo Hong

    2015-01-01

    Twin and family studies suggest that political attitudes are partially determined by an individual's genotype. The dopamine D4 receptor gene (DRD4) exon III repeat region that has been extensively studied in connection with human behaviour, is a plausible candidate to contribute to individual differences in political attitudes. A first United States study provisionally identified this gene with political attitude along a liberal–conservative axis albeit contingent upon number of friends. In a large sample of 1771 Han Chinese university students in Singapore, we observed a significant main effect of association between the DRD4 exon III variable number of tandem repeats and political attitude. Subjects with two copies of the 4-repeat allele (4R/4R) were significantly more conservative. Our results provided evidence for a role of the DRD4 gene variants in contributing to individual differences in political attitude particularly in females and more generally suggested that associations between individual genes, and neurochemical pathways, contributing to traits relevant to the social sciences can be provisionally identified. PMID:26246555

  11. Association between the dopamine D4 receptor gene exon III variable number of tandem repeats and political attitudes in female Han Chinese.

    PubMed

    Ebstein, Richard P; Monakhov, Mikhail V; Lu, Yunfeng; Jiang, Yushi; Lai, Poh San; Chew, Soo Hong

    2015-08-22

    Twin and family studies suggest that political attitudes are partially determined by an individual's genotype. The dopamine D4 receptor gene (DRD4) exon III repeat region that has been extensively studied in connection with human behaviour, is a plausible candidate to contribute to individual differences in political attitudes. A first United States study provisionally identified this gene with political attitude along a liberal-conservative axis albeit contingent upon number of friends. In a large sample of 1771 Han Chinese university students in Singapore, we observed a significant main effect of association between the DRD4 exon III variable number of tandem repeats and political attitude. Subjects with two copies of the 4-repeat allele (4R/4R) were significantly more conservative. Our results provided evidence for a role of the DRD4 gene variants in contributing to individual differences in political attitude particularly in females and more generally suggested that associations between individual genes, and neurochemical pathways, contributing to traits relevant to the social sciences can be provisionally identified.

  12. Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.

    PubMed Central

    Xie, J; Drumm, M L; Zhao, J; Ma, J; Davis, P B

    1996-01-01

    The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0

  13. Genome evolution and the evolution of exon-shuffling--a review.

    PubMed

    Patthy, L

    1999-09-30

    Recent studies on the genomes of protists, plants, fungi and animals confirm that the increase in genome size and gene number in different eukaryotic lineages is paralleled by a general decrease in genome compactness and an increase in the number and size of introns. It may thus be predicted that exon-shuffling has become increasingly significant with the evolution of larger, less compact genomes. To test the validity of this prediction, we have analyzed the evolutionary distribution of modular proteins that have clearly evolved by intronic recombination. The results of this analysis indicate that modular multidomain proteins produced by exon-shuffling are restricted in their evolutionary distribution. Although such proteins are present in all major groups of metazoa from sponges to chordates, there is practically no evidence for the presence of related modular proteins in other groups of eukaryotes. The biological significance of this difference in the composition of the proteomes of animals, fungi, plants and protists is best appreciated when these modular proteins are classified with respect to their biological function. The majority of these proteins can be assigned to functional categories that are inextricably linked to multicellularity of animals, and are of absolute importance in permitting animals to function in an integrated fashion: constituents of the extracellular matrix, proteases involved in tissue remodelling processes, various proteins of body fluids, membrane-associated proteins mediating cell-cell and cell-matrix interactions, membrane associated receptor proteins regulating cell cell communications, etc. Although some basic types of modular proteins seem to be shared by all major groups of metazoa, there are also groups of modular proteins that appear to be restricted to certain evolutionary lineages. In summary, the results suggest that exon-shuffling acquired major significance at the time of metazoan radiation. It is interesting to note that

  14. Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2β in Development

    PubMed Central

    Best, Andrew; Liu, Yilei; Jakubik, Miriam; Mende, Ylva; Ehrmann, Ingrid; Curk, Tomaz; Rossbach, Kristina; Bourgeois, Cyril F.; Stévenin, James; Grellscheid, David; Jackson, Michael S.; Wirth, Brunhilde; Elliott, David J.

    2011-01-01

    Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein. PMID:22194695

  15. Altered patterns of fractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy.

    PubMed

    Tang, Haibao; Woodhouse, Margaret R; Cheng, Feng; Schnable, James C; Pedersen, Brent S; Conant, Gavin; Wang, Xiaowu; Freeling, Michael; Pires, J Chris

    2012-04-01

    The genome sequence of the paleohexaploid Brassica rapa shows that fractionation is biased among the three subgenomes and that the least fractionated subgenome has approximately twice as many orthologs as its close (and relatively unduplicated) relative Arabidopsis than had either of the other two subgenomes. One evolutionary scenario is that the two subgenomes with heavy gene losses (I and II) were in the same nucleus for a longer period of time than the third subgenome (III) with the fewest gene losses. This "two-step" hypothesis is essentially the same as that proposed previously for the eudicot paleohexaploidy; however, the more recent nature of the B. rapa paleohexaploidy makes this model more testable. We found that subgenome II suffered recent small deletions within exons more frequently than subgenome I, as would be expected if the genes in subgenome I had already been near maximally fractionated before subgenome III was introduced. We observed that some sequences, before these deletions, were flanked by short direct repeats, a unique signature of intrachromosomal illegitimate recombination. We also found, through simulations, that short--single or two-gene--deletions appear to dominate the fractionation patterns in B. rapa. We conclude that the observed patterns of the triplicated regions in the Brassica genome are best explained by a two-step fractionation model. The triplication and subsequent mode of fractionation could influence the potential to generate morphological diversity--a hallmark of the Brassica genus.

  16. Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits.

    PubMed

    Speed, Haley E; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M; Ochoa, Christine F; Gupta, Natasha; Liu, Shunan; Powell, Craig M

    2015-07-01

    SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3(G)). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3(G/G) mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3(G/G) mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3(G/G) mouse that was engineered with such future experiments in mind.

  17. Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits.

    PubMed

    Speed, Haley E; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M; Ochoa, Christine F; Gupta, Natasha; Liu, Shunan; Powell, Craig M

    2015-07-01

    SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3(G)). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3(G/G) mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3(G/G) mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3(G/G) mouse that was engineered with such future experiments in mind. PMID:26134648

  18. Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1.

    PubMed Central

    Reisman, D; Greenberg, M; Rotter, V

    1988-01-01

    To gain insight into how transcription of the human p53 oncogene is controlled, we characterized the regulatory regions of the gene. A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5' end of the human p53 gene, as well as subfragments generated by restriction digests, was cloned upstream of the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and CAT activity was assayed in extracts of transfected cells. Two types of CAT vectors were used: Epstein-Barr virus oriP-derived constructs that were stably introduced into the human cell lines K562, Raji, and HL-60, and pSV0-CAT-derived constructs that were transiently introduced into the monkey cell line COS. By this approach we have identified two promoters for the human p53 gene. One promoter, p53P1, is located 100-250 bp upstream of the 218-bp noncoding first exon; a second, stronger promoter, p53P2, maps within the first intron. CAT activity and expression of CAT RNA indicate that p53P2 functions up to 50-fold more efficiently than p53P1. We conclude that the expression of the human p53 gene may be controlled by two promoters and that differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells. Images PMID:2839831

  19. Does natural selection explain the fine scale genetic structure at the nuclear exon Glu-5' in blue mussels from Kerguelen?

    PubMed

    Gérard, Karin; Roby, Charlotte; Bierne, Nicolas; Borsa, Philippe; Féral, Jean-Pierre; Chenuil, Anne

    2015-04-01

    The Kerguelen archipelago, isolated in the Southern Ocean, shelters a blue mussel Mytilus metapopulation far from any influence of continental populations or any known hybrid zone. The finely carved coast leads to a highly heterogeneous habitat. We investigated the impact of the environment on the genetic structure in those Kerguelen blue mussels by relating allele frequencies to habitat descriptors. A total sample comprising up to 2248 individuals from 35 locations was characterized using two nuclear markers, mac-1 and Glu-5', and a mitochondrial marker (COI). The frequency data from 9 allozyme loci in 9 of these locations were also reanalyzed. Two other nuclear markers (EFbis and EFprem's) were monomorphic. Compared to Northern Hemisphere populations, polymorphism in Kerguelen blue mussels was lower for all markers except for the exon Glu-5'. At Glu-5', genetic differences were observed between samples from distinct regions (F CT = 0.077), as well as within two regions, including between samples separated by <500 m. No significant differentiation was observed in the AMOVA analyses at the two other markers (mac-1 and COI). Like mac-1, all allozyme loci genotyped in a previous publication, displayed lower differentiation (Jost's D) and F ST values than Glu-5'. Power simulations and confidence intervals support that Glu-5' displays significantly higher differentiation than the other loci (except a single allozyme for which confidence intervals overlap). AMOVA analyses revealed significant effects of the giant kelp Macrocystis and wave exposure on this marker. We discuss the influence of hydrological conditions on the genetic differentiation among regions. In marine organisms with high fecundity and high dispersal potential, gene flow tends to erase differentiation, but this study showed significant differentiation at very small distance. This may be explained by the particular hydrology and the carved coastline of the Kerguelen archipelago, together with

  20. Does natural selection explain the fine scale genetic structure at the nuclear exon Glu-5' in blue mussels from Kerguelen?

    PubMed

    Gérard, Karin; Roby, Charlotte; Bierne, Nicolas; Borsa, Philippe; Féral, Jean-Pierre; Chenuil, Anne

    2015-04-01

    The Kerguelen archipelago, isolated in the Southern Ocean, shelters a blue mussel Mytilus metapopulation far from any influence of continental populations or any known hybrid zone. The finely carved coast leads to a highly heterogeneous habitat. We investigated the impact of the environment on the genetic structure in those Kerguelen blue mussels by relating allele frequencies to habitat descriptors. A total sample comprising up to 2248 individuals from 35 locations was characterized using two nuclear markers, mac-1 and Glu-5', and a mitochondrial marker (COI). The frequency data from 9 allozyme loci in 9 of these locations were also reanalyzed. Two other nuclear markers (EFbis and EFprem's) were monomorphic. Compared to Northern Hemisphere populations, polymorphism in Kerguelen blue mussels was lower for all markers except for the exon Glu-5'. At Glu-5', genetic differences were observed between samples from distinct regions (F CT = 0.077), as well as within two regions, including between samples separated by <500 m. No significant differentiation was observed in the AMOVA analyses at the two other markers (mac-1 and COI). Like mac-1, all allozyme loci genotyped in a previous publication, displayed lower differentiation (Jost's D) and F ST values than Glu-5'. Power simulations and confidence intervals support that Glu-5' displays significantly higher differentiation than the other loci (except a single allozyme for which confidence intervals overlap). AMOVA analyses revealed significant effects of the giant kelp Macrocystis and wave exposure on this marker. We discuss the influence of hydrological conditions on the genetic differentiation among regions. In marine organisms with high fecundity and high dispersal potential, gene flow tends to erase differentiation, but this study showed significant differentiation at very small distance. This may be explained by the particular hydrology and the carved coastline of the Kerguelen archipelago, together with

  1. Concomitant partial exon skipping by a unique missense mutation of RPS6KA3 causes Coffin-Lowry syndrome.

    PubMed

    Labonne, Jonathan D J; Chung, Min Ji; Jones, Julie R; Anand, Priya; Wenzel, Wolfgang; Iacoboni, Daniela; Layman, Lawrence C; Kim, Hyung-Goo

    2016-01-01

    Coffin-Lowry syndrome (CLS) is an X-linked semi-dominant disorder characterized by diverse phenotypes including intellectual disability, facial and digital anomalies. Loss-of-function mutations in the Ribosomal Protein S6 Kinase Polypeptide 3 (RPS6KA3) gene have been shown to be responsible for CLS. Among the large number of mutations, however, no exonic mutation causing exon skipping has been described. Here, we report a male patient with CLS having a novel mutation at the 3' end of an exon at a splice donor junction. Interestingly, this nucleotide change causes both a novel missense mutation and partial exon skipping leading to a truncated transcript. These two transcripts were identified by cDNA sequencing of RT-PCR products. In the carrier mother, we found only wildtype transcripts suggesting skewed X-inactivation. Methylation studies confirmed X-inactivation was skewed moderately, but not completely, which is consistent with her mild phenotype. Western blot showed that the mutant RSK2 protein in the patient is expressed at similar levels relative to his mother. Protein modeling demonstrated that the missense mutation is damaging and may alter binding to ATP molecules. This is the first report of exon skipping from an exonic mutation of RPS6KA3, demonstrating that a missense mutation and concomitant disruption of normal splicing contribute to the manifestation of CLS. PMID:26297997

  2. Differences among lesions with exon 19, exon 21 EGFR mutations and wild types in surgically resected non-small cell lung cancer

    PubMed Central

    Jin, Ying; Chen, Ming; Yu, Xinmin

    2016-01-01

    The clinical behavior of patients with advanced non-small cell lung cancer (NSCLC) differ between epidermal growth factor receptor (EGFR) exon 19 deletion (Ex19) and EGFR exon 21 L858R mutation (Ex21). This study aimed to evaluate whether these differences exist in surgically resected NSCLC. A total of 198 patients with surgically resected NSCLC harbouring Ex19 (n = 53), Ex21 (n = 51), and EGFR wild-type (Wt) (n = 94) were analyzed. The clinicopathological features, laboratory parameters, recurrent sites and disease-free survival (DFS) were compared according to mutational EGFR status. Ex21 occurred more frequently in female (p < 0.001), never-smokers (p < 0.001), adenocarcinoma (p < 0.001), low grade (p = 0.013) than Wt lesions. Ex19 occurred more frequently in female (p = 0.016), never-smokers (p = 0.008), adenocarcinoma (p < 0.001), low grade (p = 0.025) than Wt lesions. Ex 21 lesions (p = 0.026) had larger lepidic components than Wt lesions. Wt lesions had larger mucinous variant components than Ex21 lesions (p = 0.045) and Ex19 lesions (p = 0.015). Ex21 lesions were associated with lower pretreatment neutrophil: lymphocyte ratio (NLR) than Wt lesions (p = 0.017). The recurrent sites and DFS were similar among patients with Wt, Ex19 and Ex21. PMID:27527915

  3. Detection of TMPRSS2-ERG translocations in human prostate cancer by expression profiling using GeneChip Human Exon 1.0 ST arrays.

    PubMed

    Jhavar, Sameer; Reid, Alison; Clark, Jeremy; Kote-Jarai, Zsofia; Christmas, Timothy; Thompson, Alan; Woodhouse, Christopher; Ogden, Christopher; Fisher, Cyril; Corbishley, Cathy; De-Bono, Johann; Eeles, Rosalind; Brewer, Daniel; Cooper, Colin

    2008-01-01

    Translocation of TMPRSS2 to the ERG gene, found in a high proportion of human prostate cancer, results in overexpression of the 3'-ERG sequences joined to the 5'-TMPRSS2 promoter. The studies presented here were designed to test the ability of expression analysis on GeneChip Human Exon 1.0 ST arrays to detect 5'-TMPRSS2-ERG-3' hybrid transcripts encoded by this translocation. Monitoring the relative expression of each ERG exon revealed altered transcription of the ERG gene in 15 of a series of 27 prostate cancer samples. In all cases, exons 4 to 11 exhibited enhanced expression compared with exons 2 and 3. This pattern of expression indicated that the most abundant hybrid transcripts involve fusions to ERG exon 4, and RT-PCR analyses confirmed the joining of TMPRSS2 exon 1 to ERG exon 4 in all 15 cases. The exon expression patterns also indicated that TMPRSS2-ERG fusion transcripts commonly contain deletion of ERG exon 8. Analysis of gene-level data from the arrays allowed the identification of genes whose expression levels significantly correlated with the presence of the translocation. These studies demonstrate that expression analyses using exon arrays represent a valuable approach for detecting ETS gene translocation in prostate cancer, in parallel with analyses of gene expression profiles.

  4. Physiologically generated presenilin 1 lacking exon 8 fails to rescue brain PS1-/- phenotype and forms complexes with wildtype PS1 and nicastrin.

    PubMed

    Brautigam, Hannah; Moreno, Cesar L; Steele, John W; Bogush, Alexey; Dickstein, Dara L; Kwok, John B J; Schofield, Peter R; Thinakaran, Gopal; Mathews, Paul M; Hof, Patrick R; Gandy, Sam; Ehrlich, Michelle E

    2015-01-01

    The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1(∆exon8)). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1(∆exon8) reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1(∆exon8) is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1(∆exon8) in vivo by crossing PS1(∆exon8) transgenics with either PS1-null or Dutch APP(E693Q) mice. As a control, we crossed APP(E693Q) with mice expressing a deletion in an adjacent exon (PS1(∆exon9)). PS1(∆exon8) did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation. These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions. PMID:26608390

  5. MAGPIE/EGRET Annotation of the 2.9-Mb Drosophila melanogaster Adh Region

    PubMed Central

    Gaasterland, Terry; Sczyrba, Alexander; Thomas, Elizabeth; Aytekin-Kurban, Gulriz; Gordon, Paul; Sensen, Christoph W.

    2000-01-01

    Our challenge in annotating the 2.91-Mb Adh region of the Drosophila melanogaster genome was to identify genetic and genomic features automatically, completely, and precisely within a 6-week period. To do so, we augmented the MAGPIE microbial genome annotation system to handle eukaryotic genomic sequence data. The new configuration required the integration of eukaryotic gene-finding tools and DNA repeat tools into the automatic data collection module. It also required us to define in MAGPIE new strategies to combine data about eukaryotic exon predictions with functional data to refine the exon predictions. At the heart of the resulting new eukaryotic genome annotation system is a reverse comparison of public protein and complementary DNA sequences against the input genome to identify missing exons and to refine exon boundaries. The software modules that add eukaryotic genome annotation capability to MAGPIE are available as EGRET (Eukaryotic Genome Rapid Evaluation Tool). PMID:10779489

  6. Exonal elements and factors involved in the depolarization-induced alternative splicing of neurexin 2.

    PubMed

    Rozic, G; Lupowitz, Z; Zisapel, N

    2013-05-01

    The neurexin genes (NRXN1, NRXN2, and NRXN3) encode polymorphic presynaptic proteins that are implicated in synaptic plasticity and memory processing. In rat brain neurons grown in culture, depolarization induces reversible, calcium-dependent, repression of NRXN2α exon 11 (E11) splicing. Using Neuro2a cells as a model, we explored E11 cis elements and trans-acting factors involved in alternative splicing of NRXN2α E11 pre-mRNA under basal and depolarization conditions. E11 mutation studies revealed two motifs, CTGCCTG (enhancer) and GCACCCA (suppressor) regulating NRXN2α E11 alternative splicing. Subsequent E11 RNA affinity pull-down experiments demonstrated heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP L binding to this exon. Under depolarization, the amount of E11-bound hnRNP L (but not of hnRNP K) increased, in parallel to NRXN2α E11 splicing repression. Depletion of hnRNP K or hnRNP L in the Neuro2a cells by specific siRNAs enhanced NRXN2α E11 splicing and ablated the depolarization-induced repression of this exon. In addition, depolarization suppressed whereas hnRNP K depletion enhanced NRXN2α expression. These results indicate a role for hnRNP K in regulation of NRXN2α expression and of hnRNP L in the activity-dependent alternative splicing of neurexins which may potentially govern trans-synaptic signaling required for memory processing.

  7. Normal phenotype in conditional androgen receptor (AR) exon 3-floxed neomycin-negative male mice.

    PubMed

    Rana, Kesha; Clarke, Michele V; Zajac, Jeffrey D; Davey, Rachel A; MacLean, Helen E

    2014-01-01

    Androgens (testosterone and dihydrotestosterone) acting via the androgen receptor (AR) are required for male sexual differentiation, and also regulate the development of many other tissues including muscle, fat and bone. We previously generated an AR(lox) mouse line with exon 3 of the AR gene targeted by loxP sites. The deletion of exon 3 is in-frame, so only the DNA binding-dependent actions of the AR are deleted, but non-DNA binding-dependent actions are retained. This line also contained an antibiotic resistance selection cassette, neomycin (neo) in intron 3, which was also flanked by loxP sites. Hemizygous AR(lox) male mice demonstrated a phenotype of hyperandrogenization, with increased mass of androgen-dependent tissues. We hypothesized that this hyperandrogenization was likely to be due to the presence of the neo cassette. In this study, we have generated an AR(lox) neo-negative mouse line, using the EIIa-cre deleter mouse line to remove the neo cassette. Hemizygous AR(lox) neo-negative male mice have a normal phenotype, with normal body mass and normal mass of androgen-dependent tissues including the testis, seminal vesicles, kidney, spleen, heart and retroperitoneal fat. This neo-negative exon 3-targeted mouse line is the only floxed AR mouse line available to study the DNA binding-dependent actions of the AR in a tissue-specific manner, and is suitable for investigation in all tissues. This study demonstrates the importance of removing the selection cassette, which can potentially alter the phenotype of floxed mouse lines even in the absence of detectable effects on target gene expression.

  8. Sequencing based typing for genetic polymorphisms in exons, 2, 3 and 4 of the MICA gene.

    PubMed

    Katsuyama, Y; Ota, M; Ando, H; Saito, S; Mizuki, N; Kera, J; Bahram, S; Nose, Y; Inoko, H

    1999-08-01

    We have established a sequencing based typing (SBT) method for detection of genetic polymorphism in the exon 2 to 4 domains of the major histocompatibility complex (MHC) class I chain-related gene A (MICA) and applied it to allele typing of 130 healthy Japanese individuals. A 2.2-kb segment including exons 2, 3 and 4 of the MICA gene was amplified by a pair of generic primers followed by cycle sequencing using exon-specific nested primers. In total, 8 alleles were observed in a Japanese population and the most frequent allele was MICA008 with the gene frequency of 30.8%. MICA009 was the second most frequent (16.5%), while the rarest one was MICA007 (1.2%). MICA alleles displayed strong linkage equilibria with HLA-B antigens (i.e. MICA008 with B7, B48, B60 and B61; MICA009 with B51 and B52; MICA002 with B35, B39, B58 and B67; MICA004 with B44, MICA007 with B13 and B27; MICA010 with B46, B62 and B48, MICA012 with B54, B55, B56 and B59; MICA019 and B70, B71 and B62). Recently, the B48 haplotype has been reported to lack the entire MICA gene by a large-scale deletion in a Japanese population. Among 8 serologically B48 homozygous individuals, 4 were found to represent this MICA null allele as assessed by no polymerase chain reaction (PCR) amplification using MICA-specific primers, while the remaining four possessed the intact MICA gene with MICA008 or MICA010.

  9. Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

    PubMed

    Gabreski, Nicole A; Vaghasia, Janki K; Novakova, Silvia S; McDonald, Neil Q; Pierchala, Brian A

    2016-07-29

    Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function.

  10. Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

    PubMed Central

    King, Robert; Bird, Nicholas; Ramirez-Gonzalez, Ricardo; Coghill, Jane A.; Patil, Archana; Hassani-Pak, Keywan; Uauy, Cristobal; Phillips, Andrew L.

    2015-01-01

    Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues. PMID:26335335

  11. Colocalisation of predicted exonic splicing enhancers in BRCA2 with reported sequence variants.

    PubMed

    Pettigrew, Christopher A; Wayte, Nicola; Wronski, Ania; Lovelock, Paul K; Spurdle, Amanda B; Brown, Melissa A

    2008-07-01

    Disruption of the breast cancer susceptibility gene BRCA2 is associated with increased risk of developing breast and ovarian cancer. Over 1800 sequence changes in BRCA2 have been reported, although for many the pathogenicity is unclear. Classifying these changes remains a challenge, as they may disrupt regulatory sequences as well as the primary protein coding sequence. Sequence changes located in the splice site consensus sequences often disrupt splicing, however sequence changes located within exons are also able to alter splicing patterns. Unfortunately, the presence of these exonic splicing enhancers (ESEs) and the functional effect of variants within ESEs it is currently difficult to predict. We have previously developed a method of predicting which sequence changes within exons are likely to affect splicing, using BRCA1 as an example. In this paper, we have predicted ESEs in BRCA2 using the web-based tool ESEfinder and incorporated the same series of filters (increased threshold, 125 nt limit and evolutionary conservation of the motif) in order to identify predicted ESEs that are more likely to be functional. Initially 1114 ESEs were predicted for BRCA2, however after all the filters were included, this figure was reduced to 31, 3% of the original number of predicted ESEs. Reported unclassified sequence variants in BRCA2 were found to colocalise to 55% (17/31) of these conserved ESEs, while polymorphisms colocalised to 0 of the conserved ESEs. In summary, we have identified a subset of unclassified sequence variants in BRCA2 that may adversely affect splicing and thereby contribute to BRCA2 disruption.

  12. Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy.

    PubMed

    Ottenheijm, Coen A C; Buck, Danielle; de Winter, Josine M; Ferrara, Claudia; Piroddi, Nicoletta; Tesi, Chiara; Jasper, Jeffrey R; Malik, Fady I; Meng, Hui; Stienen, Ger J M; Beggs, Alan H; Labeit, Siegfried; Poggesi, Corrado; Lawlor, Michael W; Granzier, Henk

    2013-06-01

    Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by

  13. Novel SNP identification in exon 3 of HSP90AA1 gene and their association with heat tolerance traits in Karan Fries (Bos taurus × Bos indicus) cows under tropical climatic condition.

    PubMed

    Kumar, Rakesh; Gupta, Ishwar Dayal; Verma, Archana; Singh, Sohan Vir; Verma, Nishant; Vineeth, M R; Magotra, Ankit; Das, Ramendra

    2016-04-01

    Heat shock proteins (HSPs) act as molecular chaperones those are preferentially transcribed in respose to heat stress and the polymorphism in HSP genes associated with heat tolerance traits in cows. HSP90AA1 gene has been mapped on Bos taurus autosome 21 (BTA-21) and spans nearly 5368 bp comprising of 11 exons out of which the first exon does not translate. The present study was done on Karan Fries (5/8 HF × 3/8 Tharparkar) cows reared in tropical climate with the objectives of identifying single-nucleotide polymorphisms (SNPs) in targeted regions (exon 3) of HSP90AA1 gene and analyzing their association with heat tolerance traits in Karan Fries cows. Respiration rate (RR) and rectal temperature (RT) were recorded once daily for four consecutive days during probable extreme hours in different seasons or temperature humidity index (THI), viz., winter, spring, and summer. For detecting single-nucleotide polymorphisms, sequence data were analyzed using BioEdit software (version 7.2). Comparative sequence analysis of HSP90AA1 gene showed point mutation, viz., g.1209A>G (exon 3) as compared to Bos taurus (NCBI Ref Seq: AC_000178.1). Association analysis indicated that THI was influenced (P < 0.01) by RR, RT, and HTC. Similarly, SNPs at locus g.1209A>G were categorized into three genotypes, i.e., AA, AG, and GG, and the least squares means (LSMEANS) of RR, RT, and HTC for GG (homozygous) genotype were significantly lower (P < 0.01) than AA (homozygous) and AG (heterozygous) genotypes. These findings may partly suggest that cows with GG genotypes were favored for heat tolerance trait, which can be used as an aid to selection for thermo-tolerance Karan Fries cows for better adaptation in subtropical and tropical hot climate. PMID:26898694

  14. African Cattle do not Carry Unique Mutations on the Exon 9 of the ARHGAP15 Gene.

    PubMed

    Álvarez, Isabel; Pérez-Pardal, Lucía; Traoré, Amadou; Fernández, Iván; Goyache, Félix

    2016-01-01

    A panel of 81 Asian, African and European cattle (Bos taurus and B. indicus) was sequenced for the exon 9 of the ARHGAP15, a strong candidate for cattle trypanotolerance on BTA2. The analyses provided five different haplotypes defined by four (two nonsynonymous) mutations. Neutrality tests suggest a recent sweep in the studied bovine sequences. The two most frequent haplotypes (H1 and H3) gathered 88% of the chromosomes analyzed and were present in all the cattle groups analyzed, including Asian zebu and European cattle. The current results question the sole association of the polymorphism identified, including mutation c.53317501A > C, with the trypanotolerant response in West African cattle.

  15. WISCOD: a statistical web-enabled tool for the identification of significant protein coding regions.

    PubMed

    Vilardell, Mireia; Parra, Genis; Civit, Sergi

    2014-01-01

    Classically, gene prediction programs are based on detecting signals such as boundary sites (splice sites, starts, and stops) and coding regions in the DNA sequence in order to build potential exons and join them into a gene structure. Although nowadays it is possible to improve their performance with additional information from related species or/and cDNA databases, further improvement at any step could help to obtain better predictions. Here, we present WISCOD, a web-enabled tool for the identification of significant protein coding regions, a novel software tool that tackles the exon prediction problem in eukaryotic genomes. WISCOD has the capacity to detect real exons from large lists of potential exons, and it provides an easy way to use global P value called expected probability of being a false exon (EPFE) that is useful for ranking potential exons in a probabilistic framework, without additional computational costs. The advantage of our approach is that it significantly increases the specificity and sensitivity (both between 80% and 90%) in comparison to other ab initio methods (where they are in the range of 70-75%). WISCOD is written in JAVA and R and is available to download and to run in a local mode on Linux and Windows platforms. PMID:25313355

  16. WISCOD: a statistical web-enabled tool for the identification of significant protein coding regions.

    PubMed

    Vilardell, Mireia; Parra, Genis; Civit, Sergi

    2014-01-01

    Classically, gene prediction programs are based on detecting signals such as boundary sites (splice sites, starts, and stops) and coding regions in the DNA sequence in order to build potential exons and join them into a gene structure. Although nowadays it is possible to improve their performance with additional information from related species or/and cDNA databases, further improvement at any step could help to obtain better predictions. Here, we present WISCOD, a web-enabled tool for the identification of significant protein coding regions, a novel software tool that tackles the exon prediction problem in eukaryotic genomes. WISCOD has the capacity to detect real exons from large lists of potential exons, and it provides an easy way to use global P value called expected probability of being a false exon (EPFE) that is useful for ranking potential exons in a probabilistic framework, without additional computational costs. The advantage of our approach is that it significantly increases the specificity and sensitivity (both between 80% and 90%) in comparison to other ab initio methods (where they are in the range of 70-75%). WISCOD is written in JAVA and R and is available to download and to run in a local mode on Linux and Windows platforms.

  17. Wilson's disease caused by alternative splicing and Alu exonization due to a homozygous 3039-bp deletion spanning from intron 1 to exon 2 of the ATP7B gene.

    PubMed

    Mameli, Eva; Lepori, Maria Barbara; Chiappe, Francesca; Ranucci, Giusy; Di Dato, Fabiola; Iorio, Raffaele; Loudianos, Georgios

    2015-09-15

    We describe a case of Wilson's disease (WD) diagnosed at 5 years after routine biochemical test showed increased aminotransferases. Mutation analysis of the ATP7B gene revealed a 3039-bp deletion in the homozygous state spanning from the terminal part of intron 1 to nt position 368 of exon 2. This deletion results in the activation of 3 cryptic splice sites: an AG acceptor splice site in nt positions 578-579 producing a different breakpoint and removing the first 577 nts of exon 2, an acceptor and a donor splice site in nt positions 20363-4 and 20456-7, respectively, in intron 1, resulting in the activation of a 94-bp cryptic Alu exon being incorporated into the mature transcript. The resulting alternative transcript contains a TAG stop codon in the first amino acid position of the cryptic exon, likely producing a truncated, non-functional protein. This study shows that intron exonization can also occur in humans through naturally occurring gross deletions. The results suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counseling and diagnosis of WD. Moreover these studies help to better establish new molecular mechanisms producing Wilson's disease.

  18. Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

    PubMed Central

    van der Klift, Heleen M; Jansen, Anne M L; van der Steenstraten, Niki; Bik, Elsa C; Tops, Carli M J; Devilee, Peter; Wijnen, Juul T

    2015-01-01

    A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants. PMID:26247049

  19. Exonic versus intronic SNPs: contrasting roles in revealing the population genetic differentiation of a widespread bird species

    PubMed Central

    Zhan, X; Dixon, A; Batbayar, N; Bragin, E; Ayas, Z; Deutschova, L; Chavko, J; Domashevsky, S; Dorosencu, A; Bagyura, J; Gombobaatar, S; Grlica, I D; Levin, A; Milobog, Y; Ming, M; Prommer, M; Purev-Ochir, G; Ragyov, D; Tsurkanu, V; Vetrov, V; Zubkov, N; Bruford, M W

    2015-01-01

    Recent years have seen considerable progress in applying single nucleotide polymorphisms (SNPs) to population genetics studies. However, relatively few have attempted to use them to study the genetic differentiation of wild bird populations and none have examined possible differences of exonic and intronic SNPs in these studies. Here, using 144 SNPs, we examined population genetic differentiation in the saker falcon (Falco cherrug) across Eurasia. The position of each SNP was verified using the recently sequenced saker genome with 108 SNPs positioned within the introns of 10 fragments and 36 SNPs in the exons of six genes, comprising MHC, MC1R and four others. In contrast to intronic SNPs, both Bayesian clustering and principal component analyses using exonic SNPs consistently revealed two genetic clusters, within which the least admixed individuals were found in Europe/central Asia and Qinghai (China), respectively. Pairwise D analysis for exonic SNPs showed that the two populations were significantly differentiated and between the two clusters the frequencies of five SNP markers were inferred to be influenced by selection. Central Eurasian populations clustered in as intermediate between the two main groups, consistent with their geographic position. But the westernmost populations of central Europe showed evidence of demographic isolation. Our work highlights the importance of functional exonic SNPs for studying population genetic pattern in a widespread avian species. PMID:25074575

  20. Exonic versus intronic SNPs: contrasting roles in revealing the population genetic differentiation of a widespread bird species.

    PubMed

    Zhan, X; Dixon, A; Batbayar, N; Bragin, E; Ayas, Z; Deutschova, L; Chavko, J; Domashevsky, S; Dorosencu, A; Bagyura, J; Gombobaatar, S; Grlica, I D; Levin, A; Milobog, Y; Ming, M; Prommer, M; Purev-Ochir, G; Ragyov, D; Tsurkanu, V; Vetrov, V; Zubkov, N; Bruford, M W

    2015-01-01

    Recent years have seen considerable progress in applying single nucleotide polymorphisms (SNPs) to population genetics studies. However, relatively few have attempted to use them to study the genetic differentiation of wild bird populations and none have examined possible differences of exonic and intronic SNPs in these studies. Here, using 144 SNPs, we examined population genetic differentiation in the saker falcon (Falco cherrug) across Eurasia. The position of each SNP was verified using the recently sequenced saker genome with 108 SNPs positioned within the introns of 10 fragments and 36 SNPs in the exons of six genes, comprising MHC, MC1R and four others. In contrast to intronic SNPs, both Bayesian clustering and principal component analyses using exonic SNPs consistently revealed two genetic clusters, within which the least admixed individuals were found in Europe/central Asia and Qinghai (China), respectively. Pairwise D analysis for exonic SNPs showed that the two populations were significantly differentiated and between the two clusters the frequencies of five SNP markers were inferred to be influenced by selection. Central Eurasian populations clustered in as intermediate between the two main groups, consistent with their geographic position. But the westernmost populations of central Europe showed evidence of demographic isolation. Our work highlights the importance of functional exonic SNPs for studying population genetic pattern in a widespread avian species. PMID:25074575

  1. Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm

    PubMed Central

    Doran, Philip; Wilton, Steve D.; Fletcher, Sue; Ohlendieck, Kay

    2009-01-01

    The disintegration of the dystrophin-glycoprotein complex represents the initial pathobiochemical insult in Duchenne muscular dystrophy. However, secondary changes in signalling, energy metabolism and ion homeostasis are probably the main factors that eventually cause progressive muscle wasting. Thus, for the proper evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscles. Antisense oligomer-mediated exon skipping promises functional restoration of the primary deficiency in dystrophin. In this study, an established phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide was employed for the specific removal of exon 23 in the mutated mouse dystrophin gene transcript. Using DIGE analysis, we could show the reversal of secondary pathobiochemical abnormalities in the dystrophic diaphragm following exon-23 skipping. In analogy to the restoration of dystrophin, β-dystroglycan and neuronal nitric oxide synthase, the muscular dystrophy-associated differential expression of calsequestrin, adenylate kinase, aldolase, mitochondrial creatine kinase and cvHsp was reversed in treated muscle fibres. Hence, the re-establishment of Dp427 coded by the transcript missing exon 23 has counter-acted dystrophic alterations in Ca2+-handling, nucleotide metabolism, bioenergetic pathways and cellular stress response. This clearly establishes the exon-skipping approach as a realistic treatment strategy for diminishing diverse downstream alterations in dystrophinopathy. PMID:19132684

  2. Identification of the uncommon allele HLA-A*7403 in a Caucasian renal transplant cadaveric donor: extension of the exon 4 sequence.

    PubMed

    Canossi, A; Del Beato, T; Piazza, A; Liberatore, G; Ozzella, G; Tessitore, A; Adorno, D

    2007-06-01

    This report describes the unknown exon 4 sequence of the rare A*7403 allele, identified in a Caucasian renal transplant cadaveric donor from Italy. This sequence is identical to that of the only known A*7401 exon 4, and this result allowed us to confirm the hypothesis of the generation of A*7403 allele from the ancestor A*7402 by point mutation in exon 2.

  3. rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution.

    PubMed Central

    Wolter, F P; Fritz, C C; Willmitzer, L; Schell, J; Schreier, P H

    1988-01-01

    Five genes of the rbcS gene family of Solanum tuberosum (potato) were studied. One of these is a cDNA clone; the other four are located on two genomic clones representing two different chromosomal loci containing one (locus 1) and three genes (locus 2), respectively. The intron/exon structure of the three genes in locus 2 is highly conserved with respect to size and position. These genes contain two introns, whereas the gene from locus 1 contains three introns. Although in most cases the amino acid sequences in the transit peptide part of different rbcS genes from the same species varied considerably more than the corresponding mature amino acid sequences, one exception found in tomato and potato indicates that the transit peptide of rbcS could have a special function. A comparison of the rbcS genes of higher plants with those of prokaryotes offers suggestive evidence that introns first served as spacer material in the process of exon shuffling and then were removed stepwise during the evolution of higher plants. PMID:3422467

  4. Growth hormone receptor exon 3 isoforms and their implication in growth disorders and treatment.

    PubMed

    Jorge, Alexander A L; Arnhold, Ivo J P

    2009-04-01

    Human recombinant growth hormone (hGH) has been used to treat short stature in several different conditions, but considerable inter-individual variation in short- and long-term growth response exists. Pharmacogenomics can provide important insights into hGH therapy. The GH receptor (GHR) is the first key molecule mediating GH action. In the past 3 years, a common GHR polymorphism reflecting the presence (GHRfl) or absence (GHRd3) of exon 3 has been under intensive investigation regarding its influence on the response to hGH therapy. Studies that evaluated response to GH treatment determined by these two GHR isoforms in children with GH deficiency, girls with Turner syndrome, children born small for gestational age and patients with acromegaly showed that patients carrying the GHRd3 allele demonstrated a greater GH sensitivity than patients homozygous for the GHRfl allele. Other studies presented contradictory data, however, which may be caused by confounding factors such as small sample sizes and differences in experimental design. This GHR exon 3 genotype is the first identified genetic factor found to modulate the individual response to GH therapy. This article reviews the historical aspects and pharmacogenetic studies published to date in relation to this GHR polymorphism. The analyses of present and future validation studies may define the use of this and other polymorphisms in clinical practice, moving from pharmacogenetics to routine application and allowing individualization of hGH doses to optimize final outcome.

  5. Modeling Exon-Specific Bias Distribution Improves the Analysis of RNA-Seq Data.

    PubMed

    Liu, Xuejun; Zhang, Li; Chen, Songcan

    2015-01-01

    RNA-seq technology has become an important tool for quantifying the gene and transcript expression in transcriptome study. The two major difficulties for the gene and transcript expression quantification are the read mapping ambiguity and the overdispersion of the read distribution along reference sequence. Many approaches have been proposed to deal with these difficulties. A number of existing methods use Poisson distribution to model the read counts and this easily splits the counts into the contributions from multiple transcripts. Meanwhile, various solutions were put forward to account for the overdispersion in the Poisson models. By checking the similarities among the variation patterns of read counts for individual genes, we found that the count variation is exon-specific and has the conserved pattern across the samples for each individual gene. We introduce Gamma-distributed latent variables to model the read sequencing preference for each exon. These variables are embedded to the rate parameter of a Poisson model to account for the overdispersion of read distribution. The model is tractable since the Gamma priors can be integrated out in the maximum likelihood estimation. We evaluate the proposed approach, PGseq, using four real datasets and one simulated dataset, and compare its performance with other popular methods. Results show that PGseq presents competitive performance compared to other alternatives in terms of accuracy in the gene and transcript expression calculation and in the downstream differential expression analysis. Especially, we show the advantage of our method in the analysis of low expression. PMID:26448625

  6. SinEx DB: a database for single exon coding sequences in mammalian genomes.

    PubMed

    Jorquera, Roddy; Ortiz, Rodrigo; Ossandon, F; Cárdenas, Juan Pablo; Sepúlveda, Rene; González, Carolina; Holmes, David S

    2016-01-01

    Eukaryotic genes are typically interrupted by intragenic, noncoding sequences termed introns. However, some genes lack introns in their coding sequence (CDS) and are generally known as 'single exon genes' (SEGs). In this work, a SEG is defined as a nuclear, protein-coding gene that lacks introns in its CDS. Whereas, many public databases of Eukaryotic multi-exon genes are available, there are only two specialized databases for SEGs. The present work addresses the need for a more extensive and diverse database by creating SinEx DB, a publicly available, searchable database of predicted SEGs from 10 completely sequenced mammalian genomes including human. SinEx DB houses the DNA and protein sequence information of these SEGs and includes their functional predictions (KOG) and the relative distribution of these functions within species. The information is stored in a relational database built with My SQL Server 5.1.33 and the complete dataset of SEG sequences and their functional predictions are available for downloading. SinEx DB can be interrogated by: (i) a browsable phylogenetic schema, (ii) carrying out BLAST searches to the in-house SinEx DB of SEGs and (iii) via an advanced search mode in which the database can be searched by key words and any combination of searches by species and predicted functions. SinEx DB provides a rich source of information for advancing our understanding of the evolution and function of SEGs.Database URL: www.sinex.cl.

  7. Fatty acid composition of beef is associated with exonic nucleotide variants of the gene encoding FASN.

    PubMed

    Oh, Dongyep; Lee, Yoonseok; La, Boomi; Yeo, Jungsou; Chung, Euiryong; Kim, Younyoung; Lee, Chaeyoung

    2012-04-01

    Genetic associations of fatty acid composition with exonic single nucleotide polymorphisms (SNPs) in the gene encoding fatty acid synthase (FASN) were examined using 513 Korean cattle. All five individual SNPs of g.12870 T>C, g.13126 T>C, g.15532 C>A, g.16907 T>C and g.17924 G>A were associated with a variety of fatty acid compositions and further with marbling score (P < 0.05). Their genotypes of CC, TT, AA, TT, and GG were associated with increased monounsaturated fatty acids and with decreased saturated fatty acids (P < 0.05). The genotypes at all the SNPs also increased marbling score (P < 0.05). Further genetic associations with fatty acid composition suggested that homozygous genotype with the haplotype of ATG at g.15532, g.16907, and g.17924 in a linkage disequilibrium block increased monounsaturated fatty acids and marbling score (P < 0.05). We concluded that the five exonic SNPs of g.12870, g.13126, g.15532, g.16907, and g.17924 in the FASN gene could change fatty acid contents. Their genotypes of CC, TT, AA, TT, and GG and haplotype of ATG at g.15532, g.16907, and g.17924 were recommended for genetic improvement of beef quality.

  8. MFN2 deletion of exons 7 and 8: founder mutation in the UK population.

    PubMed

    Carr, Aisling S; Polke, James M; Wilson, Jacob; Pelayo-Negro, Ana L; Laura, Matilde; Nanji, Tina; Holt, James; Vaughan, Jennifer; Rankin, Julia; Sweeney, Mary G; Blake, Julian; Houlden, Henry; Reilly, Mary M

    2015-06-01

    Mitofusin 2 (MFN2) mutations are the most common cause of axonal Charcot-Marie-Tooth disease (CMT2). The majority are inherited in an autosomal dominant manner but recessive and semi-dominant kindreds have also been described. We previously reported a deletion of exons 7 and 8 resulting in nonsense-mediated decay, segregating with disease when present in trans with another pathogenic MFN2 mutation. Detailed clinical and electrophysiological data on a series of five affected patients from four kindreds and, when available, their parents and relatives were collected. MFN2 Sanger sequencing, multiplex ligation probe amplification, and haplotype analysis were performed. A severe early-onset CMT phenotype was seen in all cases: progressive distal weakness, wasting, and sensory loss from infancy or early childhood. Optic atrophy (four of five) and wheelchair dependency in childhood were common (four of five). All were compound heterozygous for a deletion of exons 7 and 8 in MFN2 with another previously reported pathogenic mutation (Phe216Ser, Thr362Met, and Arg707Trp). Carrier parents and relatives were unaffected (age range: 24-82 years). Haplotype analysis confirmed that the deletion had a common founder in all families.

  9. Identification of point mutations in exon 2 of GDF9 gene in Kermani sheep.

    PubMed

    Khodabakhshzadeh, R; Mohammadabadi, M R; Esmailizadeh, A K; Moradi Shahrebabak, H; Bordbar, F; Ansari Namin, S

    2016-01-01

    Screening the fertile ewes from national herds to detect the major genes for prolificacy is an effective way to create the fertile flocks. Growth differentiation factor (GDF) 9 is a member of the transforming growth factor β superfamily that is essential for folliculogenesis and female fertility. The aim of this study was to detect single nucleotide polymorphisms (SNPs) in exon 2 of GDF9 gene in Kermani sheep breed using PCR-SSCP. Genomic DNA was extracted from whole blood of collected samples using salting-out method. Whole exon 2 of GDF9 gene was amplified (634 bp and 647 bp fragments) using designed specific primers. The single stranded conformation polymorphism (SSCP) patterns of PCR products were studied using electrophoresis on acrylamide gel and silver-nitrate staining method. Finally, 4 banding patterns for the first primer pair and 4 banding patterns for the second primer pair were obtained. Also, indices of population genetic per SNP were calculated using Gen Alex 6.41 software. The sequencing results showed the presence of 3 mutations (SNP) (443, 477 and 721 positions) in the studied population. PMID:27487501

  10. SinEx DB: a database for single exon coding sequences in mammalian genomes

    PubMed Central

    Jorquera, Roddy; Ortiz, Rodrigo; Ossandon, F.; Cárdenas, Juan Pablo; Sepúlveda, Rene; González, Carolina; Holmes, David S.

    2016-01-01

    Eukaryotic genes are typically interrupted by intragenic, noncoding sequences termed introns. However, some genes lack introns in their coding sequence (CDS) and are generally known as ‘single exon genes’ (SEGs). In this work, a SEG is defined as a nuclear, protein-coding gene that lacks introns in its CDS. Whereas, many public databases of Eukaryotic multi-exon genes are available, there are only two specialized databases for SEGs. The present work addresses the need for a more extensive and diverse database by creating SinEx DB, a publicly available, searchable database of predicted SEGs from 10 completely sequenced mammalian genomes including human. SinEx DB houses the DNA and protein sequence information of these SEGs and includes their functional predictions (KOG) and the relative distribution of these functions within species. The information is stored in a relational database built with My SQL Server 5.1.33 and the complete dataset of SEG sequences and their functional predictions are available for downloading. SinEx DB can be interrogated by: (i) a browsable phylogenetic schema, (ii) carrying out BLAST searches to the in-house SinEx DB of SEGs and (iii) via an advanced search mode in which the database can be searched by key words and any combination of searches by species and predicted functions. SinEx DB provides a rich source of information for advancing our understanding of the evolution and function of SEGs. Database URL: www.sinex.cl PMID:27278816

  11. Basal exon skipping and genetic pleiotropy: A predictive model of disease pathogenesis.

    PubMed

    Drivas, Theodore G; Wojno, Adam P; Tucker, Budd A; Stone, Edwin M; Bennett, Jean

    2015-06-10

    Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies). Molecular biologic examination of CEP290 transcript and protein expression in cells from patients carrying CEP290 mutations, measured by quantitative polymerase chain reaction and Western blotting, correlated with disease severity and corroborated our model. We show that basal exon skipping may be the mechanism underlying the disease pleiotropy caused by CEP290 mutations. Applying our model to a different disease gene, CC2D2A (coiled-coil and C2 domains-containing protein 2A), we found that the same correlations held true. Our model explains the phenotypic diversity of two different inherited ciliopathies and may establish a new model for the pathogenesis of other pleiotropic human diseases.

  12. The exon junction complex is required for definition and excision of neighboring introns in Drosophila

    PubMed Central

    Hayashi, Rippei; Handler, Dominik; Ish-Horowicz, David

    2014-01-01

    Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon–exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events. PMID:25081352

  13. The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

    PubMed Central

    Malone, Colin D.; Mestdagh, Claire; Akhtar, Junaid; Kreim, Nastasja; Deinhard, Pia; Sachidanandam, Ravi; Treisman, Jessica

    2014-01-01

    The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. PMID:25104425

  14. Selection preserves Ubiquitin Specific Protease 4 alternative exon skipping in therian mammals

    PubMed Central

    Vlasschaert, Caitlyn; Xia, Xuhua; Gray, Douglas A.

    2016-01-01

    Ubiquitin specific protease 4 (USP4) is a highly networked deubiquitinating enzyme with reported roles in cancer, innate immunity and RNA splicing. In mammals it has two dominant isoforms arising from inclusion or skipping of exon 7 (E7). We evaluated two plausible mechanisms for the generation of these isoforms: (A) E7 skipping due to a long upstream intron and (B) E7 skipping due to inefficient 5′ splice sites (5′SS) and/or branchpoint sites (BPS). We then assessed whether E7 alternative splicing is maintained by selective pressure or arose from genetic drift. Both transcript variants were generated from a USP4-E7 minigene construct with short flanking introns, an observation consistent with the second mechanism whereby differential splice signal strengths are the basis of E7 skipping. Optimization of the downstream 5′SS eliminated E7 skipping. Experimental validation of the correlation between 5′SS identity and exon skipping in vertebrates pinpointed the +6 site as the key splicing determinant. Therian mammals invariably display a 5′SS configuration favouring alternative splicing and the resulting isoforms have distinct subcellular localizations. We conclude that alternative splicing of mammalian USP4 is under selective maintenance and that long and short USP4 isoforms may target substrates in various cellular compartments. PMID:26833277

  15. The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina

    PubMed Central

    Murphy, Daniel; Carstens, Russ

    2016-01-01

    Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such “switch-like” exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments. PMID:27541351

  16. SinEx DB: a database for single exon coding sequences in mammalian genomes.

    PubMed

    Jorquera, Roddy; Ortiz, Rodrigo; Ossandon, F; Cárdenas, Juan Pablo; Sepúlveda, Rene; González, Carolina; Holmes, David S

    2016-01-01

    Eukaryotic genes are typically interrupted by intragenic, noncoding sequences termed introns. However, some genes lack introns in their coding sequence (CDS) and are generally known as 'single exon genes' (SEGs). In this work, a SEG is defined as a nuclear, protein-coding gene that lacks introns in its CDS. Whereas, many public databases of Eukaryotic multi-exon genes are available, there are only two specialized databases for SEGs. The present work addresses the need for a more extensive and diverse database by creating SinEx DB, a publicly available, searchable database of predicted SEGs from 10 completely sequenced mammalian genomes including human. SinEx DB houses the DNA and protein sequence information of these SEGs and includes their functional predictions (KOG) and the relative distribution of these functions within species. The information is stored in a relational database built with My SQL Server 5.1.33 and the complete dataset of SEG sequences and their functional predictions are available for downloading. SinEx DB can be interrogated by: (i) a browsable phylogenetic schema, (ii) carrying out BLAST searches to the in-house SinEx DB of SEGs and (iii) via an advanced search mode in which the database can be searched by key words and any combination of searches by species and predicted functions. SinEx DB provides a rich source of information for advancing our understanding of the evolution and function of SEGs.Database URL: www.sinex.cl. PMID:27278816

  17. Identification of point mutations in exon 2 of GDF9 gene in Kermani sheep.

    PubMed

    Khodabakhshzadeh, R; Mohammadabadi, M R; Esmailizadeh, A K; Moradi Shahrebabak, H; Bordbar, F; Ansari Namin, S

    2016-01-01

    Screening the fertile ewes from national herds to detect the major genes for prolificacy is an effective way to create the fertile flocks. Growth differentiation factor (GDF) 9 is a member of the transforming growth factor β superfamily that is essential for folliculogenesis and female fertility. The aim of this study was to detect single nucleotide polymorphisms (SNPs) in exon 2 of GDF9 gene in Kermani sheep breed using PCR-SSCP. Genomic DNA was extracted from whole blood of collected samples using salting-out method. Whole exon 2 of GDF9 gene was amplified (634 bp and 647 bp fragments) using designed specific primers. The single stranded conformation polymorphism (SSCP) patterns of PCR products were studied using electrophoresis on acrylamide gel and silver-nitrate staining method. Finally, 4 banding patterns for the first primer pair and 4 banding patterns for the second primer pair were obtained. Also, indices of population genetic per SNP were calculated using Gen Alex 6.41 software. The sequencing results showed the presence of 3 mutations (SNP) (443, 477 and 721 positions) in the studied population.

  18. The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina.

    PubMed

    Murphy, Daniel; Cieply, Benjamin; Carstens, Russ; Ramamurthy, Visvanathan; Stoilov, Peter

    2016-08-01

    Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such "switch-like" exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments. PMID:27541351

  19. Exon skipping and translation in patients with frameshift deletions in the dystrophin gene

    SciTech Connect

    Sherratt, T.G.; Dubowitz, V.; Sewry, C.A.; Strong, P.N. ); Vulliamy, T. )

    1993-11-01

    Although many Duchenne muscular dystrophy patients have a deletion in the dystrophin gene which disrupts the translational reading frame, they express dystrophin in a small proportion of skeletal muscle fibers ([open quotes]revertant fibers[close quotes]). Antibody studies have shown, indirectly, that dystrophin synthesis in revertant fibers is facilitated by a frame-restoring mechanism; in the present study, the feasibility of mRNA splicing was investigated. Dystrophin transcripts were analyzed in skeletal muscle from individuals possessing revertant fibers and a frameshift deletion in the dystrophin gene. In each case a minor in-frame transcript was detected, in which exons adjacent to those deleted from the genome had been skipped. There appeared to be some correlation between the levels of in-frame transcripts and the predicted translation products. Low levels of alternatively spliced transcripts were also present in normal muscle. The results provide further evidence of exon skipping in the dystrophin gene and indicate that this may be involved in the synthesis of dystrophin by revertant fibers. 44 refs., 12 figs.

  20. [Genetic characteristics on exon 4 of prolactin gene in 12 water buffalo populations].

    PubMed

    Yuan, Feng; Miao, Yong-Wang; Li, Da-Lin; Tang, Shou-Kun; Xv, Zheng; Huo, Jin-Long; Qi, Hong

    2010-12-01

    The prolactin exerts obvious adjustment and control function for mammary gland development, lactation and milk protein gene expression in water buffalo. In this study the sequence features and polymorphisms of the exon 4 in prolactin gene were examined in 385 individuals which came from 12 river and swamp type buffalo populations using DNA direct sequencing and PCR-SSCP methods. The results showed that the sequence of exon 4 in prolactin gene was consists of 180 nucleotides, the fragment had high conservative character in different species. The e4. 109 C>T substitution was detected in nine swamp buffalo populations, and it was a silent mutation and was not associated with the traits of milk yield in buffalo. The PBA gene was the predominant gene in seven swamp type buffalo populations, while PBB gene was the dominant gene in Dehong and Fuzhong populations. The frequencies of PBA in swamp type buffalo was 0.400 -0.917 and the average value was 0.629+/-0.049. The polymorphism wasn't found in river buffalo, all the samples from river buffalo were holding nucleotides e4.109 C. The results indicate that there is distinct genetic differentiation between swamp and river type buffalo.

  1. Exalign: a new method for comparative analysis of exon-intron gene structures.

    PubMed

    Pavesi, Giulio; Zambelli, Federico; Caggese, Corrado; Pesole, Graziano

    2008-05-01

    The evolution of genes is usually studied and reconstructed at the sequence level, that is, by comparing and aligning their genomic, transcript or protein sequences. However, including the exon-intron structure of genes in the analysis can provide further and useful information, for example to draw reliable phylogenetic relationships left unsolved by traditional sequence-based evolutionary studies, or to shed further light on patterns of intron gain and loss. In spite of this, no tool especially devised for this task is currently available. In this work we present Exalign, an algorithm designed to retrieve, compare and search for the exon-intron structure of existing gene annotations, that has been implemented in a software tool freely accessible through a web interface as well as available for download. We present different applications of our method, from the reconstruction of the evolutionary history of homologous gene families to the detection of as of today unknown cases of intron loss in human and rodents, and, remarkably, two never reported intron gain events in human and mouse. The web interface for accessing Exalign is available at http://www.pesolelab.it/exalign/ or http://www.beacon.unimi.it/exalign/

  2. Direct identification of all oncogenic mutants in KRAS exon 1 by cycling temperature capillary electrophoresis.

    PubMed

    Bjørheim, Jens; Gaudernack, Gustav; Giercksky, Karl-Erik; Ekstrøm, Per O

    2003-01-01

    Over the past few decades, advances in genetics and molecular biology have revolutionized our understanding of cancer initiation and progression. Molecular progression models outlining genetic events have been developed for many solid tumors, including colon cancer. Previous reports in the literature have shown a relationship between different KRAS mutations and prognosis and response to medical treatment in colon cancer patients. Furthermore, the presence of a mutated KRAS has been correlated with different clinicopathological variables including age and gender of patients and tumor location. To our knowledge, few institutions screen for KRAS mutations on regular basis in colon cancer patients despite such evidence that knowledge of KRAS exon 1 status is informative. Here, we report on a mutation analysis method adapted to a 96-capillary electrophoresis instrument that allows identification of all 12 oncogenic mutations in KRAS exon 1 under denaturing conditions. To determine the optimal parameters, a series of DNA constructs generated by site-directed mutagenesis was analyzed and the migration times of all mutant peaks were measured. A classification tree was then made based on the differences in migration time between the mutants and an internal standard. A randomized series of 500 samples constructed with mutagenesis as well as 60 blind samples from sporadic colon carcinomas was analyzed to test the method. No wild-type samples were scored as mutants and all mutants were correctly identified. Post polymerase chain reaction (PCR) analysis time of 96 samples was performed within 40 min. PMID:12652573

  3. Modeling Exon-Specific Bias Distribution Improves the Analysis of RNA-Seq Data

    PubMed Central

    Liu, Xuejun; Zhang, Li; Chen, Songcan

    2015-01-01

    RNA-seq technology has become an important tool for quantifying the gene and transcript expression in transcriptome study. The two major difficulties for the gene and transcript expression quantification are the read mapping ambiguity and the overdispersion of the read distribution along reference sequence. Many approaches have been proposed to deal with these difficulties. A number of existing methods use Poisson distribution to model the read counts and this easily splits the counts into the contributions from multiple transcripts. Meanwhile, various solutions were put forward to account for the overdispersion in the Poisson models. By checking the similarities among the variation patterns of read counts for individual genes, we found that the count variation is exon-specific and has the conserved pattern across the samples for each individual gene. We introduce Gamma-distributed latent variables to model the read sequencing preference for each exon. These variables are embedded to the rate parameter of a Poisson model to account for the overdispersion of read distribution. The model is tractable since the Gamma priors can be integrated out in the maximum likelihood estimation. We evaluate the proposed approach, PGseq, using four real datasets and one simulated dataset, and compare its performance with other popular methods. Results show that PGseq presents competitive performance compared to other alternatives in terms of accuracy in the gene and transcript expression calculation and in the downstream differential expression analysis. Especially, we show the advantage of our method in the analysis of low expression. PMID:26448625

  4. Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy.

    PubMed

    Bornert, Olivier; Kühl, Tobias; Bremer, Jeroen; van den Akker, Peter C; Pasmooij, Anna Mg; Nyström, Alexander

    2016-08-01

    Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB. PMID:27157667

  5. 46 CFR 193.15-16 - Lockout valves.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... lockout valve must be provided on any carbon dioxide extinguishing system protecting a space over 6,000... complete isolation of the system from the protected space or spaces, making it impossible for carbon... the valve is locked open at all times, except while maintenance is being performed on...

  6. Imaging Sciences Workshop, Proceedings, November 15-16, 1995

    SciTech Connect

    Candy, J.V.

    1995-11-01

    Welcome to the Imaging Sciences Workshop sponsored by C.A.S.I.S., the Center for Advanced Signal & Image Sciences. Many programs at LLNL use advanced signal and image processing techniques, and the Center was established to encourage the exchange of ideas and to promote collaboration by individuals from these programs. This Workshop is an opportunity for LLNL personnel and invited speakers from other organizations not only to present new work, but, perhaps more importantly, to discuss problems in an informal and friendly setting. This year marks the opening of the CASIS Reference Library in Building 272, and we encourage all attendees to stop by for a look and to make use of it in the future. The Technical Program covers a wide variety of applications at LLNL including physical systems for collecting data and processing techniques for recovering and enhancing images. We hope that you enjoy the presentations, and we encourage you to participate in the discussions. Thanks for attending.

  7. Exon 11 skipping of SCN10A coding for voltage-gated sodium channels in dorsal root ganglia.

    PubMed

    Schirmeyer, Jana; Szafranski, Karol; Leipold, Enrico; Mawrin, Christian; Platzer, Matthias; Heinemann, Stefan H

    2014-01-01

    The voltage-gated sodium channel Na(V)1.8 (encoded by SCN10A) is predominantly expressed in dorsal root ganglia(DRG) and plays a critical role in pain perception. We analyzed SCN10A transcripts isolated from human DRGs using deep sequencing and found a novel splice variant lacking exon 11, which codes for 98 amino acids of the domain I/II linker. Quantitative PCR analysis revealed an abundance of this variant of up to 5–10% in human, while no such variants were detected in mouse or rat. Since no obvious functional differences between channels with and without the exon-11 sequence were detected, it is suggested that SCN10A exon 11 skipping in humans is a tolerated event. PMID:24763188

  8. Exon 24-25 deletion of RB1 in a four-generation low-penetrance retinoblastoma family

    SciTech Connect

    Du, D.; Gallie, B.L.; Mostachfi, H.

    1994-09-01

    The majority of RB1 mutations that lead to retinoblastoma result in absent of truncated protein, deleted for the domains shown to be important in binding of the protein to transcription factors and to viral transforming proteins. Promoter and missense mutations have been identified in the uncommon retinoblastoma families with low penetrance and expressivity. We have found an exon 24-25 deletion of RB1 in a large family with four affected generations but low penetrance (only 50% of deletion-carriers develop tumors), expressivity (only 30% of affected are bilateral), and one member with retinoma. The deletion was found by screening of exons in genomic DNA using quantitative PCR amplification comparing to a control chromosome and sample monosomic, diploid and trisomic for RB1. Prenatal diagnosis was possible based on recognition of the deletion. Since this deletion is sufficient to cause retinoblastoma, these exons must be important to the tumor suppressor function of the protein.

  9. Exon-focused genome-wide association study of obsessive-compulsive disorder and shared polygenic risk with schizophrenia

    PubMed Central

    Costas, J; Carrera, N; Alonso, P; Gurriarán, X; Segalàs, C; Real, E; López-Solà, C; Mas, S; Gassó, P; Domènech, L; Morell, M; Quintela, I; Lázaro, L; Menchón, J M; Estivill, X; Carracedo, Á

    2016-01-01

    Common single-nucleotide polymorphisms (SNPs) account for a large proportion of the heritability of obsessive-compulsive disorder (OCD). Co-ocurrence of OCD and schizophrenia is commoner than expected based on their respective prevalences, complicating the clinical management of patients. This study addresses two main objectives: to identify particular genes associated with OCD by SNP-based and gene-based tests; and to test the existence of a polygenic risk shared with schizophrenia. The primary analysis was an exon-focused genome-wide association study of 370 OCD cases and 443 controls from Spain. A polygenic risk model based on the Psychiatric Genetics Consortium schizophrenia data set (PGC-SCZ2) was tested in our OCD data. A polygenic risk model based on our OCD data was tested on previous data of schizophrenia from our group. The most significant association at the gene-based test was found at DNM3 (P=7.9 × 10−5), a gene involved in synaptic vesicle endocytosis. The polygenic risk model from PGC-SCZ2 data was strongly associated with disease status in our OCD sample, reaching its most significant value after removal of the major histocompatibility complex region (lowest P=2.3 × 10−6, explaining 3.7% of the variance). The shared polygenic risk was confirmed in our schizophrenia data. In conclusion, DNM3 may be involved in risk to OCD. The shared polygenic risk between schizophrenia and OCD may be partially responsible for the frequent comorbidity of both disorders, explaining epidemiological data on cross-disorder risk. This common etiology may have clinical implications. PMID:27023174

  10. Shared Gene Structures and Clusters of Mutually Exclusive Spliced Exons within the Metazoan Muscle Myosin Heavy Chain Genes

    PubMed Central

    Kollmar, Martin; Hatje, Klas

    2014-01-01

    Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times

  11. Carbonic anhydrase II deficiency: Single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients

    SciTech Connect

    Hu, P.Y.; Ernst, A.R.; Sly, W.S. ); Venta, P.J. ); Skaggs, L.A.; Tashian, R.E. )

    1994-04-01

    To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. The authors report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation. These findings identify a novel mutation common to Hispanic patients from the Caribbean islands and provide a ready means for PCR-based diagnosis of the [open quotes]Hispanic mutation.[close quotes] The basis for their phenotypic variability is not yet clear. 15 refs., 5 figs., 1 tab.

  12. Exon-focused genome-wide association study of obsessive-compulsive disorder and shared polygenic risk with schizophrenia.

    PubMed

    Costas, J; Carrera, N; Alonso, P; Gurriarán, X; Segalàs, C; Real, E; López-Solà, C; Mas, S; Gassó, P; Domènech, L; Morell, M; Quintela, I; Lázaro, L; Menchón, J M; Estivill, X; Carracedo, Á

    2016-01-01

    Common single-nucleotide polymorphisms (SNPs) account for a large proportion of the heritability of obsessive-compulsive disorder (OCD). Co-ocurrence of OCD and schizophrenia is commoner than expected based on their respective prevalences, complicating the clinical management of patients. This study addresses two main objectives: to identify particular genes associated with OCD by SNP-based and gene-based tests; and to test the existence of a polygenic risk shared with schizophrenia. The primary analysis was an exon-focused genome-wide association study of 370 OCD cases and 443 controls from Spain. A polygenic risk model based on the Psychiatric Genetics Consortium schizophrenia data set (PGC-SCZ2) was tested in our OCD data. A polygenic risk model based on our OCD data was tested on previous data of schizophrenia from our group. The most significant association at the gene-based test was found at DNM3 (P=7.9 × 10(-5)), a gene involved in synaptic vesicle endocytosis. The polygenic risk model from PGC-SCZ2 data was strongly associated with disease status in our OCD sample, reaching its most significant value after removal of the major histocompatibility complex region (lowest P=2.3 × 10(-6), explaining 3.7% of the variance). The shared polygenic risk was confirmed in our schizophrenia data. In conclusion, DNM3 may be involved in risk to OCD. The shared polygenic risk between schizophrenia and OCD may be partially responsible for the frequent comorbidity of both disorders, explaining epidemiological data on cross-disorder risk. This common etiology may have clinical implications. PMID:27023174

  13. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

    PubMed Central

    Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2016-01-01

    The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases. PMID:26761715

  14. Gene search in the FSHD region on 4q35

    SciTech Connect

    Deutekom, J.C.T. van; Romberg, S.; Geel, M. van

    1994-09-01

    In the search for the FSHD gene on 4q35, four overlapping cosmids spanning a region of 95 kb including the deletion-prone repeated units were subcloned as well as subjected to cDNA selection and exon trap strategies. A total of 300 selected clones with an average length of 500 bp were mapped back to the cosmids. None of the clones appeared to be single copy. Sequence data of most clones and the related genomic regions were compared. cDNA clones with a high homolgy (>90%) and a low repetitive hybridization pattern were further analyzed by Zoo- and Northern blotting and by sequence analysis programs like GRAIL. Excellent and good exons could be identified and some clones showed evolutionary conservation. With the best cDNA, genomic and exon trap clones, several cDNA libraries were screened. The obtained cDNAs identified different genes, none of which originated from 4q35. 3{prime} RACE experiments were performed using primers derived of predicted exons especially in a 2.2 kb EcoRI fragment about 20 kb centromeric of the repeats. So far, only non-4q35 genes could be identified. Altogether, our results support other recent studies indicating that the FSHD gene is most likely not encoded by the 3.3 kb repeated units. Moreover, the region centromeric of these repeats appeared to contain abundant repetitive sequences and homologies to several other chromosomes, complicating the identification of the FSHD gene.

  15. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    SciTech Connect

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N.

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  16. De novo exon 1 missense mutations of SKI and Shprintzen-Goldberg syndrome: two new cases and a clinical review.

    PubMed

    Au, P Y Billie; Racher, Hilary E; Graham, John M; Kramer, Nancy; Lowry, R Brian; Parboosingh, Jillian S; Innes, A Micheil

    2014-03-01

    Shprintzen-Goldberg syndrome (OMIM #182212) is a connective tissue disorder characterized by craniosynostosis, distinctive craniofacial features, skeletal abnormalities, marfanoid body habitus, aortic dilatation, and intellectual disability. Mutations in exon 1 of SKI have recently been identified as being responsible for approximately 90% of reported individuals diagnosed clinically with Shprintzen-Goldberg syndrome. SKI is a known regulator of TGFβ signaling. Therefore, like Marfan syndrome and Loeys-Dietz syndrome, Shprintzen-Goldberg syndrome is likely caused by deregulated TGFβ signals, explaining the considerable phenotypic overlap between these three disorders. We describe two additional patients with exon 1 SKI mutations and review the clinical features and literature of Shprintzen-Goldberg syndrome.

  17. Definition of the human N-myc promoter region during development in a transgenic mouse model.

    PubMed

    Tai, K F; Rogers, S W; Pont-Kingdon, G; Carroll, W L

    1999-09-01

    The N-myc oncogene directs organogenesis, and gene amplification is associated with aggressive forms of neuroblastoma, a common malignant tumor in children. N-myc is expressed in fetal epithelium, and expression decreases markedly postnatally. To localize sequences responsible for directing expression, we have analyzed the human N-myc promoter. We noted previously that N-myc promoter regions 5' to exon 1 directed reporter gene expression in all cell lines, including those without detectable N-myc transcripts. However, when promoter constructs included 3' exon 1 and the 5' portion of intron 1, reporter activity was detected only when there was expression of the endogenous gene. To determine the role of this "tissue-specific region" in directing expression during development, we generated transgenic mice carrying N-myc promoter lacZ minigenes that contained 5' N-myc promoter elements alone or the promoter linked to the 3' exon 1/5' intron 1 tissue-specific region. Animals lacking the tissue-specific exon 1/intron 1 region showed beta-galactosidase expression in the CNS, but expression was not observed in other organs in which endogenously derived N-myc transcripts were seen. Within the CNS, transgene expression was seen mainly in the olfactory system and was not observed in other areas in which expression of the murine gene has been noted. In contrast, no transgene expression was observed in any of the animals carrying the tissue-specific exon 1/intron 1 region. Thus, sequences that direct expression within the olfactory system were contained within our 5' promoter transgene, whereas sequences that guide the ubiquitous expression of N-myc during organogenesis lie outside the regions studied here. Finally, the exon 1/intron 1 region seems to act in a dominant fashion to repress expression in the CNS from the immediate 5' N-myc promoter. PMID:10473038

  18. Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy.

    PubMed

    Verheul, Ruurd C; van Deutekom, Judith C T; Datson, Nicole A

    2016-01-01

    Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0-100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD. PMID:27612288

  19. Polymorphism and haplotype analyses of swine leukocyte antigen DQA exons 2, 3, 4, and their associations with piglet diarrhea in Chinese native pig.

    PubMed

    Huang, X Y; Yang, Q L; Yuan, J H; Gun, S B

    2015-01-01

    In this study, 290 Chinese native Yantai black pig piglets were investigated to identify gene polymorphisms, for haplotype reconstruction, and to determine the association between piglet diarrhea and swine leukocyte antigen (SLA) class II DQA exons 2, 3, and 4 by polymerase chain reaction-single stranded conformational polymorphism and cloning sequencing. The results showed that the 5, 8, and 7 genotypes were identified from SLA-DQA exon 2, 3, and 4, respectively, based on the single-stranded conformational polymorphism banding patterns and found a novel allele D in exon 2 and 2 novel mutational sites of allele C (c.4828T>C) and allele F (c.4617T>C) in exon 3. Polymorphism information content testing showed that exon 2 was moderately polymorphic and that exons-3 and -4 loci were highly polymorphic. The piglet diarrhea scores for genotypes AB (1.40 ± 0.14) and AC (1.54 ± 0.17) in exon 2, AA (1.22 ± 0.32), BC (1.72 ± 0.13), DD (1.67 ± 0.35), and CF (1.22 ± 0.45) in exon 3, and AD (2.35 ± 0.25) in exon 4 were significantly higher than those for the other genotypes (P ≤ 0.05) in DQA exons. There were 14 reconstructed haplotypes in the 3 exons from 290 individuals and Hap12 may be the diarrhea-resistant gene. Haplotype distribution was extremely uneven, and the SLA-DQA gene showed genetic linkage. In this study, we identified molecular genetic markers and provided a theoretical foundation for future pig anti-disease resistance breeding. PMID:26400277

  20. Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy

    PubMed Central

    Verheul, Ruurd C.; van Deutekom, Judith C. T.; Datson, Nicole A.

    2016-01-01

    Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0–100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD. PMID:27612288

  1. Polymorphism and haplotype analyses of swine leukocyte antigen DQA exons 2, 3, 4, and their associations with piglet diarrhea in Chinese native pig.

    PubMed

    Huang, X Y; Yang, Q L; Yuan, J H; Gun, S B

    2015-09-08

    In this study, 290 Chinese native Yantai black pig piglets were investigated to identify gene polymorphisms, for haplotype reconstruction, and to determine the association between piglet diarrhea and swine leukocyte antigen (SLA) class II DQA exons 2, 3, and 4 by polymerase chain reaction-single stranded conformational polymorphism and cloning sequencing. The results showed that the 5, 8, and 7 genotypes were identified from SLA-DQA exon 2, 3, and 4, respectively, based on the single-stranded conformational polymorphism banding patterns and found a novel allele D in exon 2 and 2 novel mutational sites of allele C (c.4828T>C) and allele F (c.4617T>C) in exon 3. Polymorphism information content testing showed that exon 2 was moderately polymorphic and that exons-3 and -4 loci were highly polymorphic. The piglet diarrhea scores for genotypes AB (1.40 ± 0.14) and AC (1.54 ± 0.17) in exon 2, AA (1.22 ± 0.32), BC (1.72 ± 0.13), DD (1.67 ± 0.35), and CF (1.22 ± 0.45) in exon 3, and AD (2.35 ± 0.25) in exon 4 were significantly higher than those for the other genotypes (P ≤ 0.05) in DQA exons. There were 14 reconstructed haplotypes in the 3 exons from 290 individuals and Hap12 may be the diarrhea-resistant gene. Haplotype distribution was extremely uneven, and the SLA-DQA gene showed genetic linkage. In this study, we identified molecular genetic markers and provided a theoretical foundation for future pig anti-disease resistance breeding.

  2. The complete exon-intron structure of the 156-kb human gene NFKB1, which encodes the p105 and p50 proteins of transcription factors NF-{kappa}B and I{kappa}B-{gamma}: Implications for NF-{kappa}B-mediated signal transduction

    SciTech Connect

    Heron, E.; Deloukas, P.; van Loon, A.P.G.M.

    1995-12-10

    The NFKB1 gene encodes three proteins of the NF-{kappa}/Rel and I{kappa}B families: p105, p50, and (in mouse) I{kappa}B-{gamma}. We determined the complete genomic structure of human NFKB1. NFKB1 spans 156 kb and has 24 exons with introns varying between 40,000 and 323 bp in length. Although NFKB2, which encodes p100 and p52, also has 24 exons and has a comparable exon-intron structure, it is 20 times shorter than NFKB1. We propose that the long size of NFKB1 is important for transient activation of NF-{kappa}B complexes containing p50. I{kappa}B-{gamma} corresponds to the carboxyl-terminal half of p105. DNA sequence analysis showed that the 3{prime}-end of human intron 11 and the 5{prime}-end of exon 12 of NFKB1 are colinear with the 5{prime}-untranslated region of mouse I{kappa}B-{gamma} cDNA. I{kappa}B-{gamma} is thus likely to be generated by transcription starting within intron 11 and not by alternative splicing of the mouse mRNA encoding p105 and p50. 71 refs., 5 figs., 1 tab.

  3. Function and Pathological Implications of Exon Junction Complex Factor Y14

    PubMed Central

    Chuang, Tzu-Wei; Lee, Kou-Ming; Tarn, Woan-Yuh

    2015-01-01

    Eukaryotic mRNA biogenesis involves a series of interconnected steps, including nuclear pre-mRNA processing, mRNA export, and surveillance. The exon-junction complex (EJC) is deposited on newly spliced mRNAs and coordinates several downstream steps of mRNA biogenesis. The EJC core protein, Y14, functions with its partners in nonsense-mediated mRNA decay and translational enhancement. Y14 plays additional roles in mRNA metabolism, some of which are independent of the EJC, and it is also involved in other cellular processes. Genetic mutations or aberrant expression of Y14 results in physiological abnormality and may cause disease. Therefore, it is important to understand the various functions of Y14 and its physiological and pathological roles. PMID:25866920

  4. Precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons

    SciTech Connect

    Lemaire, H.G.; Kang, J.; Mueller-Hill, B. ); Salbaum, J.M.; Multhaup, G.; Beyreuther, K. ); Bayney, R.M.; Unterbeck, A. )

    1989-01-25

    Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4{sub 695}) of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.

  5. Adhesion domain of human T11 (CD2) is encoded by a single exon.

    PubMed Central

    Richardson, N E; Chang, H C; Brown, N R; Hussey, R E; Sayre, P H; Reinherz, E L

    1988-01-01

    The 50-kDa T11 (CD2) T-lymphocyte surface glycoprotein facilitates physical adhesion between T-lineage cells and their cognate cellular counterparts (cytotoxic T-lymphocytes-target cells, helper T lymphocytes-antigen-presenting cells, or thymocytes-thymic epithelium) as well as signaling through the antigen-specific T3-Ti receptor complex. To examine the relationship between the structure and function of the T11 molecule, we have utilized a baculoviral expression system to produce milligram quantities of the hydrophilic extracellular T11 segment. Enzyme cleavage, microsequencing, and HPLC analyses of the expressed protein in conjunction with genomic cloning information show that the domain involved in cellular adhesion is encoded by a single 321-base-pair exon. Images PMID:2455894

  6. Quantitative visualization of alternative exon expression from RNA-seq data

    PubMed Central

    Katz, Yarden; Wang, Eric T.; Silterra, Jacob; Schwartz, Schraga; Wong, Bang; Thorvaldsdóttir, Helga; Robinson, James T.; Mesirov, Jill P.; Airoldi, Edoardo M.; Burge, Christopher B.

    2015-01-01

    Motivation: Analysis of RNA sequencing (RNA-Seq) data revealed that the vast majority of human genes express multiple mRNA isoforms, produced by alternative pre-mRNA splicing and other mechanisms, and that most alternative isoforms vary in expression between human tissues. As RNA-Seq datasets grow in size, it remains challenging to visualize isoform expression across multiple samples. Results: To help address this problem, we present Sashimi plots, a quantitative visualization of aligned RNA-Seq reads that enables quantitative comparison of exon usage across samples or experimental conditions. Sashimi plots can be made using the Broad Integrated Genome Viewer or with a stand-alone command line program. Availability and implementation: Software code and documentation freely available here: http://miso.readthedocs.org/en/fastmiso/sashimi.html Contact: mesirov@broadinstitute.org, airoldi@fas.harvard.edu or cburge@mit.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25617416

  7. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

    PubMed Central

    Tompkins, Joshua D.; Jung, Marc; Chen, Chang-yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

    2016-01-01

    The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors. PMID:26981572

  8. The exon junction complex as a node of post-transcriptional networks.

    PubMed

    Le Hir, Hervé; Saulière, Jérôme; Wang, Zhen

    2016-01-01

    The exon junction complex (EJC) is deposited onto mRNAs following splicing and adopts a unique structure, which can both stably bind to mRNAs and function as an anchor for diverse processing factors. Recent findings revealed that in addition to its established roles in nonsense-mediated mRNA decay, the EJC is involved in mRNA splicing, transport and translation. While structural studies have shed light on EJC assembly, transcriptome-wide analyses revealed differential EJC loading at spliced junctions. Thus, the EJC functions as a node of post-transcriptional gene expression networks, the importance of which is being revealed by the discovery of increasing numbers of EJC-related disorders. PMID:26670016

  9. Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance.

    PubMed Central

    Desai, A. J.; Luqmani, Y. A.; Walters, J. E.; Coope, R. C.; Dagg, B.; Gomm, J. J.; Pace, P. E.; Rees, C. N.; Thirunavukkarasu, V.; Shousha, S.; Groome, N. P.; Coombes, R.; Ali, S.

    1997-01-01

    A variant form of the human oestrogen receptor (ER) mRNA lacking sequences encoded within exon 5 has been described (Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malloy BW, McGuire WL 1991, Cancer Res 51: 105-109). We have examined the expression of the exon 5-deleted ER (HE delta5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT - PCR). HE delta5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% CI, P=0.05). Presence of the HE delta5 mRNA was associated with the presence of immunohistochemically detected ER (P=0.015) and progesterone receptor (PR) (P=0.02). There was a positive correlation between the presence of HE delta5 and disease-free survival (P=0.05), suggesting that the presence of HE delta5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HE delta5. This antibody recognized the variant but not the wild-type ER protein. We show that HE delta5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HE delta5 in mammalian cells and s