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Sample records for exonic region 15-16

  1. Agora XI: The Learning Region (Thessaloniki, Greece, March 15-16, 2001). CEDEFOP Panorama Series.

    ERIC Educational Resources Information Center

    Guggenheim, Eric Fries, Ed.

    This conference program describes "education and training agencies taking a leading role in promoting innovation on a regional basis" as the focus of the Agora and puts forward the following three key issues to be debated during it: (1) the concept of the learning region itself and a critique of its strengths and weaknesses; (2)…

  2. 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

    2015-01-07

    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.

  3. Evidence for a novel exon in the coding region of the adenomatous polyposis coli (APC) gene

    SciTech Connect

    Xia, Ling; St. Denis, K.A.; Bapat, B.

    1995-08-10

    Germline mutations of the tumor suppressor gene APC cause familial adenomatous polyposis. Somatic APC alterations are involved in several sporadic neoplasma, including colorectal, duodenal, gastric, and esophageal carcinoma. The APC mRNA is encoded by 15 exons. Additional transcripts have been reported, due to alternative splicing of coding as well as noncoding regions. Two mRNA isoforms occur due to a deletion of exon 7 or a partial deletion of exon 9. We have identified a novel exon, flanked by APC exons 10 and 11, which is expressed as an alternatively transcribed product of the gene. Further, we have shown that the novel exon consists of a heptad repeat motif and is conserved across species. 18 refs., 2 figs.

  4. A Fast Algorithm for Exonic Regions Prediction in DNA Sequences

    PubMed Central

    Saberkari, Hamidreza; Shamsi, Mousa; Heravi, Hamed; Sedaaghi, Mohammad Hossein

    2013-01-01

    The main purpose of this paper is to introduce a fast method for gene prediction in DNA sequences based on the period-3 property in exons. First, the symbolic DNA sequences were converted to digital signal using the electron ion interaction potential method. Then, to reduce the effect of background noise in the period-3 spectrum, we used the discrete wavelet transform at three levels and applied it on the input digital signal. Finally, the Goertzel algorithm was used to extract period-3 components in the filtered DNA sequence. The proposed algorithm leads to decrease the computational complexity and hence, increases the speed of the process. Detection of small size exons in DNA sequences, exactly, is another advantage of the algorithm. The proposed algorithm ability in exon prediction was compared with several existing methods at the nucleotide level using: (i) specificity - sensitivity values; (ii) receiver operating curves (ROC); and (iii) area under ROC curve. Simulation results confirmed that the proposed method can be used as a promising tool for exon prediction in DNA sequences. PMID:24672762

  5. Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

    PubMed

    Newton, Derek C; Bevan, Sian C; Choi, Stephen; Robb, G Brett; Millar, Adam; Wang, Yang; Marsden, Philip A

    2003-01-03

    Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

  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. 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.

  8. Association study of the CNR1 gene exon 3 alternative promoter region polymorphisms and substance dependence

    PubMed Central

    Herman, Aryeh I.; Kranzler, Henry R.; Cubells, Joseph F.; Gelernter, Joel; Covault, Jonathan

    2008-01-01

    An alternative promoter producing a novel 5′-untranslated region of cannabinoid receptor mRNA has recently been described in CNR1, the gene encoding the cannabinoid receptor protein. Single nucleotide polymorphisms (SNPs) adjacent to this site were reported to be associated with polysubstance abuse (Zhang et al. 2004). We examined the association of 4 SNPs (rs6928499, rs806379, rs1535255, rs2023239) in the distal region of intron 2 of CNR1 both with individual substance dependence diagnoses (i.e., alcohol, cocaine, and opioids), as well as with polysubstance dependence. The study samples consisted of European American and African American subjects with drug and or alcohol dependence (n=895), and controls (n = 472). Subjects were grouped as polysubstance dependent, opioid dependent, cocaine dependent, cannabis dependent and alcohol dependent. There was a modest association of marker rs1535255 with alcohol dependence, respectively (P=0.04), though with correction for multiple phenotype comparisons, this effect was not considered statistically significant. These findings fail to replicate the original report of an association between SNPs adjacent to an alternative CNR1 exon 3 transcription start site and polysubstance abuse. PMID:16741937

  9. Alternative exon-encoding regions of Locusta migratoria muscle myosin modulate the pH dependence of ATPase activity.

    PubMed

    Li, J; Lu, Z; He, J; Chen, Q; Wang, X; Kang, L; Li, X-D

    2016-12-01

    Whereas the vertebrate muscle myosin heavy chains (MHCs) are encoded by a family of Mhc genes, most insects examined to date contain a single Mhc gene and produce all of the different MHC isoforms by alternative RNA splicing. Here, we found that the migratory locust, Locusta migratoria, has one Mhc gene, which contains 41 exons, including five alternative exclusive exons and one differently included penultimate exon, and potentially encodes 360 MHC isoforms. From the adult L. migratoria, we identified 14 MHC isoforms (including two identical isoforms): four from flight muscle (the thorax dorsal longitudinal muscle), three from jump muscle (the hind leg extensor tibiae muscle) and seven from the abdominal intersegmental muscle. We purified myosins from flight muscle and jump muscle and characterized their motor activities. At neutral pH, the flight and the jump muscle myosins displayed similar levels of in vitro actin-gliding activity, whereas the former had a slightly higher actin-activated ATPase activity than the latter. Interestingly, the pH dependences of the actin-activated ATPase activity of these two myosins are different. Because the dominant MHC isoforms in these two muscles are identical except for the two alternative exon-encoding regions, we propose that these two alternative regions modulate the pH dependence of L. migratoria muscle myosin.

  10. 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.

  11. Haplotype determination of the upstream regulatory region and the second exon of the BoLA-DRB3 gene in Holstein cattle.

    PubMed

    Goszczynski, D E; Ripoli, M V; Takeshima, S-N; Baltian, L; Aida, Y; Giovambattista, G

    2014-03-01

    Polymorphisms of the BoLA-DRB3 gene are located primarily in the second exon [antigen binding site (ABS)] and, to a lesser extent, in the upstream regulatory region (URR). It can be hypothesised that exon 2 and the URR are under different types of natural selection. The aim of this work was to determine the URR-exon 2 haplotypes; 34 Holstein samples were genotyped by direct sequencing. A total of 7 URR alleles and 23 exon 2 alleles were detected, and 3 of the URR alleles were novel. Our results may suggest that no relationship exists between the URR and exon 2 of the BoLA-DRB3 gene (linkage disequilibrium P value > 0.05), most likely due to recombination over time. Our results also suggest that both regions of class II genes may be included in the development of new genotyping methods based on next-generation DNA sequencing technologies.

  12. Interaction of the Transcription Start Site Core Region and Transcription Factor YY1 Determine Ascorbate Transporter SVCT2 Exon 1a Promoter Activity

    PubMed Central

    Qiao, Huan; May, James M.

    2012-01-01

    Transcription of the ascorbate transporter, SVCT2, is driven by two distinct promoters in exon 1 of the transporter sequence. The exon 1a promoter lacks a classical transcription start site and little is known about regulation of promoter activity in the transcription start site core (TSSC) region. Here we present evidence that the TSSC binds the multifunctional initiator-binding protein YY1. Electrophoresis shift assays using YY1 antibody showed that YY1 is present as one of two major complexes that specifically bind to the TSSC. The other complex contains the transcription factor NF-Y. Mutations in the TSSC that decreased YY1 binding also impaired the exon 1a promoter activity despite the presence of an upstream activating NF-Y/USF complex, suggesting that YY1 is involved in the regulation of the exon 1a transcription. Furthermore, YY1 interaction with NF-Y and/or USF synergistically enhanced the exon 1a promoter activity in transient transfections and co-activator p300 enhanced their synergistic activation. We propose that the TSSC plays a vital role in the exon 1a transcription and that this function is partially carried out by the transcription factor YY1. Moreover, co-activator p300 might be able to synergistically enhance the TSSC function via a “bridge” mechanism with upstream sequences. PMID:22532872

  13. 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...

  14. 46 CFR 193.15-16 - Lockout valves.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 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...

  15. 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

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

    PubMed

    Milne, Bruce F

    2014-12-07

    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.

  17. Coding region variant 186H/R in Exon 4 of APOBEC3G among individuals of Western India.

    PubMed

    Singh, Hariom; Marathe, Shruti; Nain, Sumitra; Nema, Vijay; Angadi, Mansa; Bapat, Shradha; Pawar, Jyoti; Ghate, Manisha; Sahay, Seema; Gangakhedkar, Raman R

    2016-05-01

    The allelic variations in the AIDS restriction genes have been associated with the acquisition of HIV-1 and its progression. The distribution of antiviral gene variants significantly differs between populations. Therefore, we aimed to evaluate the distribution of variant allele of 186H/R in exon4 of APOBEC3G between HIV infected individuals and healthy controls among western Indian.In the present cross-sectional study, we enrolled a total of 153 HIV-infected patients confirmed and 156 unrelated healthy individuals. Polymorphism for 186H/R in exon4 of APOBEC3G gene was genotyped by PCR-RFLP. With the frequency of 186HR heterozygous genotype of APOBEC3G was found to be 13% in healthy controls and none in HIV infected cases. The frequency of 186HH common genotype of APOBEC3G was observed higher in HIV infected individuals compared with healthy controls (100% vs 91.7%). The variant genotype 186RR in APOBEC3G was not found in both the groups. The frequency of 186R allele of APOBEC3G was found 4.16% in healthy controls and nil in HIV-infected cases. The frequency of 186H allele of APOBEC3G was found to be higher in HIV-infected cases compared with healthy controls (100% vs 95.83%). The frequency of 186R allele in exon4 of APOBEC3G was found to be 4.16% in healthy controls. This observation differs from the previous report published from North India stating the absence of 186R allele of APOBEC3G in the North Indian individuals. The variant 186H/R in exon4 of APOBEC3G was neither associated with risk of acquisition of HIV-1 nor its progression.

  18. Region-specific expression and hormonal regulation of the first exon variants of rat prolactin receptor mRNA in rat brain and anterior pituitary gland.

    PubMed

    Nogami, H; Hoshino, R; Ogasawara, K; Miyamoto, S; Hisano, S

    2007-08-01

    Recent studies have revealed the occurrence of five first exon variants of the rat prolactin receptor mRNA, suggesting that multiple promoters direct prolactin receptor transcription in response to different regulatory factors. In the present study, regional expression of these first exon variants, as well as two prolactin receptor subtypes generated by alternative splicing, was examined in the brains and anterior pituitary glands of female rats. Expression of the long-form was detected in the choroid plexus, hypothalamus, hippocampus, cerebral cortex and anterior pituitary gland, whereas the short form was detected only in the choroid plexus. E1-3 mRNA, a first exon variant, was detected in the choroid plexus, hypothalamus, and anterior pituitary gland, whereas E1-4 was detected only in the choroid plexus. Other variants were not detectable by the polymerase chain reaction protocol employed in this study. Ovariectomy increased the short form in the choroid plexus and the E1-3 expression in the choroid plexus and pituitary gland, but changes in the long-form and E1-4 expression were minimal. Replacement of oestrogens and prolactin suggest that oestrogens down-regulate E1-3 expression in the choroid plexus and pituitary gland, and that the negative effect of oestrogen is mediated by prolactin in the pituitary gland. The present results revealed the region-specific promoter usage in prolactin receptor mRNA transcription, as well as the involvement of oestrogens in the regulation of E1-3 mRNA expression in the brain and pituitary gland.

  19. 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.

  20. 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.

  1. Proteins, exons and molecular evolution.

    PubMed

    Holland, S K; Blake, C C

    1987-01-01

    The discovery of the eukaryotic gene structure has prompted research into the potential relationship between protein structure and function and the corresponding exon/intron patterns. The exon shuffling hypothesis put forward by Gilbert and Blake suggests the encodement of structural and functional protein elements by exons which can recombine to create novel proteins. This provides an explanation for the relatively rapid evolution of proteins from a few primordial molecules. As the number of gene and protein structures increases, evidence of exon shuffling is becoming more apparent and examples are presented both from modern multi-domain proteins and ancient proteins. Recent work into the chemical properties and catalytic functions of RNA have led to hypotheses based upon the early existence of RNA. These theories suggest that the split gene structure originated in the primordial soup as a result of random RNA synthesis. Stable regions of RNA, or exons, were utilised as primitive enzymes. In response to selective pressures for information storage, the activity was directly transferred from the RNA enzymes or ribozymes, to proteins. These short polypeptides fused together to create larger proteins with a wide range of functions. Recent research into RNA processing and exon size, discussed in this review, provides a clearer insight into the evolutionary development of the gene and protein structure.

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

    PubMed

    Pai, Athma A; Baharian, Golshid; Pagé Sabourin, Ariane; Brinkworth, Jessica F; Nédélec, Yohann; Foley, Joseph W; Grenier, Jean-Christophe; Siddle, Katherine J; Dumaine, Anne; Yotova, Vania; Johnson, Zachary P; Lanford, Robert E; Burge, Christopher B; Barreiro, Luis B

    2016-09-01

    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.

  3. An exonic splicing silencer in the testes-specific DNA ligase III β exon

    PubMed Central

    Chew, Shern L.; Baginsky, Lysa; Eperon, Ian C.

    2000-01-01

    Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation. PMID:10606636

  4. Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

    PubMed Central

    Mitchell, P J; Carothers, A M; Han, J H; Harding, J D; Kas, E; Venolia, L; Chasin, L A

    1986-01-01

    Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream. Images PMID:3023846

  5. Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.

    PubMed

    Lubahn, D B; Brown, T R; Simental, J A; Higgs, H N; Migeon, C J; Wilson, E M; French, F S

    1989-12-01

    Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.

  6. Prominent use of distal 5′ transcription start sites and discovery of a large number of additional exons in ENCODE regions

    PubMed Central

    Denoeud, France; Kapranov, Philipp; Ucla, Catherine; Frankish, Adam; Castelo, Robert; Drenkow, Jorg; Lagarde, Julien; Alioto, Tyler; Manzano, Caroline; Chrast, Jacqueline; Dike, Sujit; Wyss, Carine; Henrichsen, Charlotte N.; Holroyd, Nancy; Dickson, Mark C.; Taylor, Ruth; Hance, Zahra; Foissac, Sylvain; Myers, Richard M.; Rogers, Jane; Hubbard, Tim; Harrow, Jennifer; Guigó, Roderic; Gingeras, Thomas R.; Antonarakis, Stylianos E.; Reymond, Alexandre

    2007-01-01

    This report presents systematic empirical annotation of transcript products from 399 annotated protein-coding loci across the 1% of the human genome targeted by the Encyclopedia of DNA elements (ENCODE) pilot project using a combination of 5′ rapid amplification of cDNA ends (RACE) and high-density resolution tiling arrays. We identified previously unannotated and often tissue- or cell-line-specific transcribed fragments (RACEfrags), both 5′ distal to the annotated 5′ terminus and internal to the annotated gene bounds for the vast majority (81.5%) of the tested genes. Half of the distal RACEfrags span large segments of genomic sequences away from the main portion of the coding transcript and often overlap with the upstream-annotated gene(s). Notably, at least 20% of the resultant novel transcripts have changes in their open reading frames (ORFs), most of them fusing ORFs of adjacent transcripts. A significant fraction of distal RACEfrags show expression levels comparable to those of known exons of the same locus, suggesting that they are not part of very minority splice forms. These results have significant implications concerning (1) our current understanding of the architecture of protein-coding genes; (2) our views on locations of regulatory regions in the genome; and (3) the interpretation of sequence polymorphisms mapping to regions hitherto considered to be “noncoding,” ultimately relating to the identification of disease-related sequence alterations. PMID:17567994

  7. Periodic sequence patterns in human exons

    SciTech Connect

    Baldi, P.; Brunak, S.; Engelbrecht, J.; Chauvin, Y.; Krogh, A.

    1995-12-31

    We analyze the sequential structure of human exons and their flanking introns by hidden Markov models. Together, models of donor site regions, acceptor site regions and flanked internal exons, show that exons -- besides the reading frame -- hold a specific periodic pattern. The pattern, which has the consensus: non-T(A/T)G and a minimal periodicity of roughly 10 nucleotides, is not a consequence of the nucleotide statistics in the three codon positions, nor of the well known nucleosome positioning signal. We discuss the relation between the pattern and other known sequence elements responsible for the intrinsic bending or curvature of DNA.

  8. Aβ1-15/16 as a potential diagnostic marker in neurodegenerative diseases.

    PubMed

    Nutu, Magdalena; Bourgeois, Philippe; Zetterberg, Henrik; Portelius, Erik; Andreasson, Ulf; Parent, Stéphane; Lipari, Francesco; Hall, Sara; Constantinescu, Radu; Hansson, Oskar; Blennow, Kaj

    2013-03-01

    Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reflect brain biochemistry. Using combined immunoprecipitation and mass spectrometry, we have shown that amyloid beta 1-15 (Aβ1-15) is produced by concerted β- and α-secretase cleavage of amyloid precursor protein (APP) and that the relative levels of Aβ1-16 in AD compared to controls are increased. Furthermore, drug-induced γ-secretase inhibition enhances the relative levels of Aβ1-15 and Aβ1-16. Here, we investigate a novel immunoassay for Aβ1-15/16 in a broad range of neurodegenerative conditions. The CSF level of Aβ1-15/16 was measured by the bead-based amplified luminescent proximity homogeneous assay (Alpha technology). Concentrations of Aβ1-15/16 were analyzed in subjects with Parkinson disease (PD; n = 90), PD with dementia (PDD) (n = 32), dementia with Lewy bodies (DLB) (n = 68), AD (n = 48), progressive supranuclear palsy (PSP) (n = 45), multiple system atrophy (MSA) (n = 46), and corticobasal degeneration (CBD) (n = 12). The detecting antibody is specific to the C-terminal epitope of Aβ15. We found that a carboxypeptidase (CPB) present in fetal bovine serum (FBS), a component of the buffers used, degrades Aβ1-16 to Aβ1-15, which is then detected by the Aβ1-15/16 assay. Significantly, lower levels of Aβ1-15/16 were detected in PD, PDD, PSP, and MSA compared to other neurodegenerative diseases and controls. Using the specific Aβ1-15/16 assay, a reliable quantification of Aβ1-15 or Aβ1-15/16 in CSF samples is obtained. We found reduced levels of Aβ1-15 in parkinsonian disease groups. The molecular mechanism behind this reduction is at present unknown.

  9. Identification and characterization of polymorphisms within the 5' flanking region, first exon and part of first intron of bovine GH gene.

    PubMed

    Ferraz, A L J; Bortolossi, J C; Curi, R A; Ferro, M I T; Ferro, J A; Furlan, L R

    2006-06-01

    The aim of the present study was to identify and characterize polymorphisms within the 5' flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental x Nelore (n = 30), Canchim x Nelore (n = 30) and Angus x Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648, AY662649 and AY662650, respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652, respectively). The allele sequences were compared with GenBank sequences (M57764, AF007750 and AH009106) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.

  10. 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.

  11. 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

  12. Advancing Student Achievement through Labor-Management Collaboration (Denver, Colorado, February 15-16, 2011)

    ERIC Educational Resources Information Center

    US Department of Education, 2011

    2011-01-01

    This paper presents the highlights of a conference entitled "Advancing Student Achievement through Labor-Management Collaboration" held in Denver, Colorado last February 15-16, 2011. This first-of-its-kind conference on labor-management collaboration is a historic effort to transform the relationships among local superintendents, school…

  13. 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…

  14. 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.

  15. PERSISTENT CURRENT EFFECT IN 15-16 T NB3SN ACCELERATOR DIPOLES AND ITS CORRECTION

    SciTech Connect

    Kashikhin, V. V.; Zlobin, A. V.

    2016-11-08

    Nb3Sn magnets with operating fields of 15-16 T are considered for the LHC Energy Doubler and a future Very High Energy pp Collider. Due to large coil volume, high critical current density and large superconducting (SC) filament size the persistent current effect is very large in Nb3Sn dipoles al low fields. This paper presents the results of analysis of the persistent current effect in the 15 T Nb3Sn dipole demonstrator being developed at FNAL, and describes different possibilities of its correction including passive SC wires, iron shims and coil geometry.

  16. 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

  17. Hardware accelerator for prediction of exons.

    PubMed

    Yusuf, Adeel; Khan, Shoab A

    2006-01-01

    Gene annotation is by nature a computationally intensive problem, as it needs to process huge data size of DNA sequences. This forces the need to look for alternate ways of implementing algorithms to predict exons. The paper presents an accelerator for indexing DNA sequences. The accelerator effectively exploits the 3-periodicity property exhibited by protein coding regions and indicates their presence in the sequence. Experimental results show superior performance compared with software-based approach for evaluating exons from DNA. The accelerator based PCI pluggable card offers a great utility to scientists and engineers actively involved in indexing DNA sequences.

  18. 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.

  19. 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.

  20. 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.

  1. The carcinogenicity of 15,16-dihydro-11-methyl-cyclopenta[a]phenanthren-17-one.

    PubMed Central

    Coombs, M. M.; Bhatt, T. S.; Young, S.

    1979-01-01

    Direct comparison of skin-tumour induction by 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (I) and by benzo[a]pyrene on mouse skin, both by repeated application or by initiation with a single dose followed by promotion with croton oil, demonstrated that these two carcinogens have similar potency. After repeated application of (I) the mean latent period for skin-tumour induction was linearly related to the logarithm of the dose over a 10-fold dose range. Under these conditions, application of the aryl-hydrocarbon-hydroxylase inhibitor 7,8-benzoflavone together with (I) inhibited tumour induction by about 40%. By contrast, in the 2-stage experiment, little effect on tumour incidence or latent period was observed when this inhibitor was applied with the single initiating dose of (I). Co-administration of the epoxide-hydratase inhibitor 1,1,1-trichloropropene oxide caused enhancement by shortening the latent period. After s.c. injection of (I) into mice, a similar number of tumours was induced on skin remote from the site of injection by promotion with corton oil begun either one week or 6 months after initiation. Gastric instillation of (I) into female rats induced mammary adenocarcinomas. PMID:526432

  2. Therapeutic exon skipping for dysferlinopathies?

    PubMed

    Aartsma-Rus, Annemieke; Singh, Kavita H K; Fokkema, Ivo F A C; Ginjaar, Ieke B; van Ommen, Gert-Jan; den Dunnen, Johan T; van der Maarel, Silvère M

    2010-08-01

    Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) currently tested in clinical trials. The aim is to reframe dystrophin transcripts using antisense oligonucleotides (AONs). These hide an exon from the splicing machinery to induce exon skipping, restoration of the reading frame and generation of internally deleted, but partially functional proteins. It thus relies on the characteristic of the dystrophin protein, which has essential N- and C-terminal domains, whereas the central rod domain is largely redundant. This approach may also be applicable to limb-girdle muscular dystrophy type 2B (LGMD2B), Myoshi myopathy (MM) and distal myopathy with anterior tibial onset (DMAT), which are caused by mutations in the dysferlin-encoding DYSF gene. Dysferlin has a function in repairing muscle membrane damage. Dysferlin contains calcium-dependent C2 lipid binding (C2) domains and an essential transmembrane domain. However, mildly affected patients in whom one or a large number of DYSF exons were missing have been described, suggesting that internally deleted dysferlin proteins can be functional. Thus, exon skipping might also be applicable as a LGMD2B, MM and DMAT therapy. In this study we have analyzed the dysferlin protein domains and DYSF mutations and have described what exons are promising targets with regard to applicability and feasibility. We also show that DYSF exon skipping seems to be as straightforward as DMD exon skipping, as AONs to induce efficient skipping of four DYSF exons were readily identified.

  3. 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.

  4. 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

  5. Computational definition of sequence motifs governing constitutive exon splicing.

    PubMed

    Zhang, Xiang H-F; Chasin, Lawrence A

    2004-06-01

    We have searched for sequence motifs that contribute to the recognition of human pre-mRNA splice sites by comparing the frequency of 8-mers in internal noncoding exons versus unspliced pseudo exons and 5' untranslated regions (5' untranslated regions [UTRs]) of transcripts of intronless genes. This type of comparison avoids the isolation of sequences that are distinguished by their protein-coding information. We classified sequence families comprising 2069 putative exonic enhancers and 974 putative exonic silencers. Representatives of each class functioned as enhancers or silencers when inserted into a test exon and assayed in transfected mammalian cells. As a class, the enhancer sequencers were more prevalent and the silencer elements less prevalent in all exons compared with introns. A survey of 58 reported exonic splicing mutations showed good agreement between the splicing phenotype and the effect of the mutation on the motifs defined here. The large number of effective sequences implied by these results suggests that sequences that influence splicing may be very abundant in pre-mRNA.

  6. 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.

  7. 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...) § 90.315 Special provisions governing use of frequencies in the 476-494 MHz band (TV Channels 15, 16... cochannel protection to TV stations on Channels 15, 16 and 17. Station operating parameters shall be...

  8. 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...) § 90.315 Special provisions governing use of frequencies in the 476-494 MHz band (TV Channels 15, 16... cochannel protection to TV stations on Channels 15, 16 and 17. Station operating parameters shall be...

  9. 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.

  10. 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…

  11. Computer Algorithms Used in Computing the Mk 15/16 Constant 0.7 ATA Oxygen Partial Pressure Decompression Tables.

    DTIC Science & Technology

    1983-01-01

    COMPUTER ALGORITHM USED IN COMPUTING THE FINAL W 15/16 CONSTANT 0.7 ATA OXYGEN PARTIAL . PERFORING ORG. REPORT MUNDER PRESSURE DECOMPRESSION TABLES 7...earlier Model Parameter Input Files bad only one subfile which could then be read and printed before an end of file is encounted and the program stops

  12. 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

  13. Exons, Introns, and DNA Thermodynamics

    NASA Astrophysics Data System (ADS)

    Carlon, Enrico; Malki, Mehdi Lejard; Blossey, Ralf

    2005-05-01

    The genes of eukaryotes are characterized by protein coding fragments, the exons, interrupted by introns, i.e., stretches of DNA which do not carry useful information for protein synthesis. We have analyzed the melting behavior of randomly selected human cDNA sequences obtained from genomic DNA by removing all introns. A clear correspondence is observed between exons and melting domains. This finding may provide new insights into the physical mechanisms underlying the evolution of genes.

  14. In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse.

    PubMed

    Aoki, Yoshitsugu; Nakamura, Akinori; Yokota, Toshifumi; Saito, Takashi; Okazawa, Hitoshi; Nagata, Tetsuya; Takeda, Shin'ichi

    2010-11-01

    A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5' and 3' splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.

  15. 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.

  16. FOXP2: novel exons, splice variants, and CAG repeat length stability.

    PubMed

    Bruce, Heather A; Margolis, Russell L

    2002-08-01

    FOXP2 is a transcription factor containing a polyglutamine tract, a zinc-finger motif, and a forkhead DNA-binding domain. The FOXP2 gene is located on 7q31. A missense mutation in the forkhead domain (exon 14) and a balanced reciprocal translocation t(5;7)(q22;q31.2) with a breakpoint between exons 3b and 4 have recently been associated with a speech and language disorder (SPCH1). The role of FOXP2 in this neurodevelopmental disorder suggests that mutations in FOXP2 could cause other neuropsychiatric disorders. To begin investigation of this possibility, we examined the genomic structure and CAG/CAA repeat region of FOXP2. We detected little polymorphism and no expansions in the FOXP2 CAG/CAA repeat in 142 individuals with progressive movement disorders. We found evidence of alternate splice variants and six previously undetected exons: three 5' untranslated exons (s1, s2, s3), two additional untranslated exons (2a and 2b) between exons 2 and 3, a translated exon (4a) between exons 4 and 5, and a longer version of exon 10 (10+) that contains an alternate stop codon and produces a truncated protein (FOXP2-S). Our results suggest that FOXP2 spans at least 603 kb of genomic DNA, more than twice the previously defined region, and provide evidence of a promoter region flanking exon s1. This demonstration of additional FOXP2 exons and splice variants should facilitate understanding of FOXP2 function and the search for additional FOXP2 mutations.

  17. Exon 44 nonsense mutation in two-Duchenne muscular dystrophy brothers detected by heteroduplex analysis.

    PubMed

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartolo, C; Mendell, J R

    1993-01-01

    Utilizing a heteroduplex method, we screened the dystrophin exon 43-45 region for point mutations, including small deletions and insertions. The method depends upon the formation of a heteroduplex between wild-type and mutant DNA PCR products. DNA specimens from one hundred and four DMD patients without detected deletions or duplications were multiplexed amplified for exons 43, 44, and 45. The PCR products were mixed with the PCR products from nonaffected controls, electrophoresed, and examined for the presence of altered mobility heteroduplex bands. An exon 44 nonsense mutation in two DMD brothers and a common intron 44 polymorphism were identified using this approach. Although the exon 44-45 region is a hotspot for deletion breakpoints, it does not appear to be prone to point mutations. The technique is extremely useful for screening several exons simultaneously and it allowed us to screen a large number of patients.

  18. Splicing of designer exons informs a biophysical model for exon definition.

    PubMed

    Arias, Mauricio A; Lubkin, Ashira; Chasin, Lawrence A

    2015-02-01

    Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3' end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs.

  19. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

    PubMed Central

    Hu, Dong Gui; McKinnon, Ross A.; Hulin, Julie-Ann; Mackenzie, Peter I.; Meech, Robyn

    2016-01-01

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE). PMID:28035996

  20. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

    PubMed

    Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

    2016-12-27

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE).

  1. Computational discovery of internal micro-exons.

    PubMed

    Volfovsky, Natalia; Haas, Brian J; Salzberg, Steven L

    2003-06-01

    Very short exons, also known as micro-exons, occur in large numbers in some eukaryotic genomes. Existing annotation tools have a limited ability to recognize these short sequences, which range in length up to 25 bp. Here, we describe a computational method for the identification of micro-exons using near-perfect alignments between cDNA and genomic DNA sequences. Using this method, we detected 319 micro-exons in 4 complete genomes, of which 224 were previously unknown, human (170), the nematode Caenorhabditis elegans (4), the fruit fly Drosophila melanogaster (14), and the mustard plant Arabidopsis thaliana (36). Comparison of our computational method with popular cDNA alignment programs shows that the new algorithm is both efficient and accurate. The algorithm also aids in the discovery of micro-exon-skipping events and cross-species micro-exon conservation.

  2. 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

  3. 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.

  4. Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

    PubMed Central

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-01-01

    The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. PMID:9410897

  5. 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.

  6. Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants.

    PubMed

    Simpson, C G; Hedley, P E; Watters, J A; Clark, G P; McQuade, C; Machray, G C; Brown, J W

    2000-03-01

    Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.

  7. 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.

  8. 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

  9. Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis.

    PubMed Central

    Harada, N; Utsumi, T; Takagi, Y

    1993-01-01

    Extensive screening of aromatase cDNA was carried out in cDNA libraries from various human tissues. The DNA sequences of all the isolated cDNA clones were identical in the region encoded by exons 2-10 of the aromatase gene. However, tissue-specific sequences, which were classified into four groups, were observed in the 5' portions of the clones corresponding to the region encoded by exon 1. All of them were also found in clones isolated from a human genomic library and mapped between exons 1 and 2 of the human aromatase gene reported previously, suggesting the presence of multiple exons 1 and promoters in the gene. Reverse transcription-PCR analyses of aromatase mRNAs in various tissues revealed that aromatase transcripts are tissue-specifically spliced by alternative use of multiple exons 1, although minor forms of the transcripts were also present in each tissue. Aromatase mRNA is spliced from 10 exons in most tissues, but from 9 exons in the prostate and from 10 or 11 exons in the placenta. This suggests that tissue-specific regulation of the aromatase gene in various tissues may be explained by alternative use of multiple exons 1 flanked with tissue-specific promoters. The alternative use of multiple exons 1 for liver transcripts was found to change developmentally. Furthermore, switch from an adipose-specific exon 1 to another type of exon 1 was observed in aromatase transcripts of adipose tissues of three of five breast cancer patients. Images Fig. 3 Fig. 4 PMID:8248245

  10. Molecular analysis of contiguous exons of the phenylalanine hydroxylase gene: identification of a new PKU mutation.

    PubMed Central

    Dianzani, I; Camaschella, C; Saglio, G; Ferrero, G B; Ramus, S; Ponzone, A; Cotton, R G

    1993-01-01

    A modified application of the chemical cleavage of mismatch (CCM) method has been used to screen three contiguous exons (exons 9, 10, and 11) of the phenylalanine hydroxylase gene in 17 Italian PKU patients. A new nonsense heterozygous C-->G transversion within exon 11 (S359X) was identified in a single patient. Only one of the four mutations previously reported in this DNA region in Caucasians was found. This lesion, IVS X-546, was detected in five of the 34 PKU alleles examined. Our results underline the versatility of the CCM method for scanning a gene for multiple mutations. Images PMID:8097261

  11. Molecular analysis of contiguous exons of the phenylalanine hydroxylase gene: identification of a new PKU mutation.

    PubMed

    Dianzani, I; Camaschella, C; Saglio, G; Ferrero, G B; Ramus, S; Ponzone, A; Cotton, R G

    1993-03-01

    A modified application of the chemical cleavage of mismatch (CCM) method has been used to screen three contiguous exons (exons 9, 10, and 11) of the phenylalanine hydroxylase gene in 17 Italian PKU patients. A new nonsense heterozygous C-->G transversion within exon 11 (S359X) was identified in a single patient. Only one of the four mutations previously reported in this DNA region in Caucasians was found. This lesion, IVS X-546, was detected in five of the 34 PKU alleles examined. Our results underline the versatility of the CCM method for scanning a gene for multiple mutations.

  12. Introns differ from exons by their redundancy

    SciTech Connect

    Popova, T.G.; Sadovskii, M.G.

    1995-10-01

    This paper is devoted to an analysis of the intrinsic structure of the gene from the point of view of the redundancy of different structural units - exons and introns. Human genes for which the exon-intron structure has been clearly established were studied. The redundancy of each exon and intron in the gene was determined. It was shown that, in human genes, introns are more redundant than exons. Redundancy is determined as the smallest length of a word (oliconucleotide) beginning with which all words in the studied nucleotide sequence are found exactly once. Mechanisms leading to the disruption of the general pattern of ratios of redundancy of exons and introns are studied. 12 refs., 3 tabs.

  13. Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene.

    PubMed

    Xu, Jin; Xu, Mingming; Hurd, Yasmin L; Pasternak, Gavril W; Pan, Ying-Xian

    2009-02-01

    Alternative splicing of the mu opioid receptor genes to create multiple mu receptor subtypes has been demonstrated in animals and humans. Previously, we identified a number of C-terminal variants in mice, rats and human, followed by several N-terminal variants associated with a new upstream exon in mice (exon 11). Behavioral studies in exon 11 knockout mice suggest an important role for the exon 11 variants in the analgesic actions of heroin and morphine-6beta-glucuronide, but not morphine or methadone. We now have identified a homologous human exon 11 and three similar human exon 11-associated variants, suggesting conservation of exon 11 and its associated variants across species. hMOR-1i has an additional 93 amino acids at the tip of the N-terminus but is otherwise identical to hMOR-1. When expressed in Chinese hamster ovary cells, the additional 93 amino acids in hMOR-1i had little effect on opioid binding, but significantly altered agonist-induced G-protein activation. hMOR-1G1 and hMOR-1G2 predicted six transmembrane domain variants, similar to those seen in mice. The regional expression of these exon 11-associated variants, as determined by RT-PCR, varied markedly, implying region-specific alternative splicing. The presence of exon 11-associated variants in humans raises questions regarding their potential role in heroin and morphine-6beta-glucuronide actions in people as they do in mice.

  14. Detection of an exon 53 polymorphism in the dystrophin gene.

    PubMed

    Prior, T W; Papp, A C; Snyder, P J; Sedra, M S

    1993-10-01

    We utilized a heteroduplex method to screen for small mutations in Duchenne muscular dystrophy patients who did not have deletions or duplications. A dystrophin exon 53 heteroduplex band was identified in 14.4% of the affected patients. Direct sequencing of the amplified product from DNA producing the heteroduplex revealed the presence of a polymorphism in the coding region. The codon for asparagine was converted from AAT to AAC.

  15. Impaired motor coordination in mice lacking the EDA exon of the fibronectin gene.

    PubMed

    Chauhan, Anil Kumar; Moretti, Federico Andrea; Iaconcig, Alessandra; Baralle, Francisco Ernesto; Muro, Andrés Fernando

    2005-06-03

    The extracellular matrix (ECM) plays an important role in the central nervous system (CNS) by modulating the migration of cells, axons and dendrites of neurons. Fibronectin (FN) is a major component of the ECM in the CNS and plays essential roles in development, cell adhesion and cell migration. Specific FN-isoforms, generated by alternative splicing at three conserved regions, the extra domain B (EDB), extra domain A (EDA) and type III homologies connecting segment (IIICS), have been shown to modulate these processes in vitro and in vivo. The inclusion of the EDA exon in the brain is highly regulated during development and aging, suggesting an important role of this exon in brain function. However, the direct role of FN-isoforms in brain function and behaviour is still obscure. Therefore, to directly assess the role of the FN-EDA exon in vivo, we have generated two mouse strains devoid of EDA exon regulated splicing in the FN gene that constitutively include (EDA(+/+)) or exclude (EDA(-/-)) the EDA exon in all tissues. Here, we show the behavioural consequences of the absence of regulated splicing of the EDA exon in the FN gene. Deletion of the EDA domain in the FN protein results in reduced motor-coordination abilities and vertical exploratory capacity, whereas mice that constitutively include the EDA domain displayed a decrease in locomotory activity in the open field (OF) test. These results strongly suggest that regulated splicing of the EDA exon is necessary for a normal function of the brain.

  16. 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.

  17. 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.

  18. Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.

    PubMed

    Aoki, Yoshitsugu; Yokota, Toshifumi; Nagata, Tetsuya; Nakamura, Akinori; Tanihata, Jun; Saito, Takashi; Duguez, Stephanie M R; Nagaraju, Kanneboyina; Hoffman, Eric P; Partridge, Terence; Takeda, Shin'ichi

    2012-08-21

    Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.

  19. Protein domains correlate strongly with exons in multiple eukaryotic genomes--evidence of exon shuffling?

    PubMed

    Liu, Mingyi; Grigoriev, Andrei

    2004-09-01

    We conducted a multi-genome analysis correlating protein domain organization with the exon-intron structure of genes in nine eukaryotic genomes. We observed a significant correlation between the borders of exons and domains on a genomic scale for both invertebrates and vertebrates. In addition, we found that the more complex organisms displayed consistently stronger exon-domain correlation, with substantially more significant correlations detected in vertebrates compared with invertebrates. Our observations concur with the principles of exon shuffling theory, including the prediction of predominantly symmetric phase of introns flanking the borders of correlating exons. These results suggest that extensive exon shuffling events during evolution significantly contributed to the shaping of eukaryotic proteomes.

  20. The patterns of p53 gene mutations differ inside and outside of exons 5-8 in breast and other cancers

    SciTech Connect

    Hartmann, A.; Blaszyk, H.; McGovern, R.M.

    1994-09-01

    Most studies of the p53 gene in tumors examine only exons 5-8. In these exons, there is a predominance of missense mutations clustered in four regions of high evolutionary conservation. We previously found 64 mutations in exons 5-8 of the p53 gene in 194 primary breast cancers. Herein, we report 18 additional mutations found by analyzing the promotor region, the first noncoding exon, and the remaining coding exons. Mutations were found in exons 4, 9, and 10, and flanking splice junctions, but not in the promotor region or in exons 1, 2, 3, and 11. Outside of exons 5-8 not a single missense mutation was found. Microdeletions and microinsertions predominate but nonsense and splice site mutations also occur. In contrast, the majority of mutations in exons 5-8 were missense changes which exclusively occurred at amino acids that were identical in all known p53 sequences which represent about 1.6 billion years of evolutionary divergence. The difference in the mutational pattern between these two regions of the p53 gene is due to a lack of missense and inframe microdeletion mutations outside exons 5-8 (p<.0001), whereas frameshift deletions and insertions, nonsense mutations and splicing defects are equally distributed over the gene. A review of the literature shows that the difference in the patterns of mutation inside and outside of exons 5-8 we have found in breast cancer is present in other types of cancers as well. These data show the importance of comparing equivalent exons when examining the pattern of p53 gene mutation in different populations. In addition, the paucity of missense mutations in breast and other cancers (even at amino acids identical throughout p53 gene evolution) indicates that at least some of the missense mutations in exons 2-4 and 9-11 result in a phenotype other than malignant transformation.

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

    SciTech Connect

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

    1989-02-01

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

  3. Naturally occurring nucleosome positioning signals in human exons and introns.

    PubMed

    Baldi, P; Brunak, S; Chauvin, Y; Krogh, A

    1996-11-08

    We describe the structural implications of a periodic pattern found in human exons and introns by hidden Markov models. We show that exons (besides the reading frame) have a specific sequential structure in the form of a pattern with triplet consensus non-T(A/T)G, and a minimal periodicity of roughly ten nucleotides. The periodic pattern is also present in intron sequences, although the strength per nucleotide is weaker. Using two independent profile methods based on triplet bendability parameters from DNase I experiments and nucleosome positioning data, we show that the pattern in multiple alignments of internal exon and intron sequences corresponds to a periodic "in phase" bending potential towards the major groove of the DNA. The nucleosome positioning data show that the consensus triplets (and their complements) have a preference for locations on a bent double helix where the major groove faces inward and is compressed. The in-phase triplets are located adjacent to GCC/GGC triplets known to have the strongest bias in their positioning on the nuclesome. Analysis of mRNA sequences encoding proteins with known tertiary structure exclude the possibility that the pattern is a consequence of the previously well-known periodicity caused by the encoding of alpha-helices in proteins. Finally, we discuss the relation between the bending potential of coding and non-coding regions and its impact on the translational positioning of nucleosomes and the recognition of genes by the transcriptional machinery.

  4. The role of germline promoters and I exons in cytokine-induced gene-specific class switch recombination.

    PubMed

    Dunnick, Wesley A; Shi, Jian; Holden, Victoria; Fontaine, Clinton; Collins, John T

    2011-01-01

    Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4-induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is <1% the wild-type level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

  5. Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human.

    PubMed

    Clark, Francis; Thanaraj, T A

    2002-02-15

    By spliced alignment of human DNA and transcript sequence data we constructed a data set of transcript-confirmed exons and introns from 2793 genes, 796 of which (28%) were seen to have multiple isoforms. We find that over one-third of human exons can translate in more than one frame, and that this is highly correlated with G+C content. Introns containing adenosine at donor site position +3 (A3), rather than guanosine (G3), are more common in low G+C regions, while the converse is true in high G+C regions. These two classes of introns are shown to have distinct lengths, consensus sequences and correlations among splice signals, leading to the hypothesis that A3 donor sites are associated with exon definition, and G3 donor sites with intron definition. Minor classes of introns, including GC-AG, U12-type GT-AG, weak, and putative AG-dependant introns are identified and characterized. Cassette exons are more prevalent in low G+C regions, while exon isoforms are more prevalent in high G+C regions. Cassette exon events outnumber other alternative events, while exon isoform events involve truncation twice as often as extension, and occur at acceptor sites twice as often as at donor sites. Alternative splicing is usually associated with weak splice signals, and in a majority of cases, preserves the coding frame. The reported characteristics of constitutive and alternative splice signals, and the hypotheses offered regarding alternative splicing and genome organization, have important implications for experimental research into RNA processing. The 'AltExtron' data sets are available at http://www.bit.uq.edu.au/altExtron/ and http://www.ebi.ac.uk/~thanaraj/altExtron/.

  6. Exon expression profiling reveals stimulus-mediated exon use in neural cells

    PubMed Central

    McKee, Adrienne E; Neretti, Nicola; Carvalho, Luis E; Meyer, Clifford A; Fox, Edward A; Brodsky, Alexander S; Silver, Pamela A

    2007-01-01

    Background: Neuronal cells respond to changes in intracellular calcium ([Ca2+]i) by affecting both the abundance and architecture of specific mRNAs. Although calcium-induced transcription and transcript variation have both been recognized as important sources of gene regulation, the interplay between these two phenomena has not been evaluated on a genome-wide scale. Results: Here, we show that exon-centric microarrays can be used to resolve the [Ca2+]i-modulated gene expression response into transcript-level and exon-level regulation. Global assessments of affected transcripts reveal modulation within distinct functional gene categories. We find that transcripts containing calcium-modulated exons exhibit enrichment for calcium ion binding, calmodulin binding, plasma membrane associated, and metabolic proteins. Additionally, we uncover instances of regulated exon use in potassium channels, neuroendocrine secretory proteins and metabolic enzymes, and demonstrate that regulated changes in exon expression give rise to distinct transcript variants. Conclusion: Our findings connect extracellular stimuli to specific exon behavior, and suggest that changes in transcript and exon abundance are reflective of a coordinated gene expression response to elevated [Ca2+]i. The technology we describe here lends itself readily to the resolution of stimulus-induced gene expression at both the transcript and exon levels. PMID:17683528

  7. Different RPGR exon ORF15 mutations in Canids provide insights into photoreceptor cell degeneration.

    PubMed

    Zhang, Qi; Acland, Gregory M; Wu, Wen X; Johnson, Jennifer L; Pearce-Kelling, Sue; Tulloch, Brian; Vervoort, Raf; Wright, Alan F; Aguirre, Gustavo D

    2002-05-01

    The canine disease, X-linked progressive retinal atrophy (XLPRA), is similar to human RP3, an X-linked form of retinitis pigmentosa, and maps to the same region in the X chromosome. Analysis of the physical map of the XLPRA and RP3 intervals shows a high degree of conservation in terms of genes and their order. We have found different mutations in exon ORF15 of the RPGR gene in two distinct mutant dog strains (XLPRA1, XLPRA2). Microdeletions resulting in a premature stop or a frameshift mutation result in very different retinal phenotypes, which are allele-specific and consistent for each mutation. The phenotype associated with the frameshift mutation in XLPRA2 is very severe and manifests during retinal development; the phenotype resulting from the XLPRA1 nonsense mutation is expressed only after normal photoreceptor morphogenesis. Splicing of RPGR mRNA transcripts in retina is complex, and either exon ORF15 or exon 19 can be a terminal exon. The retina-predominant transcript contains ORF15 as a terminal exon, and is expressed in normal and mutant retinas. The frameshift mutation dramatically alters the deduced amino acid sequence, and the protein aggregates in the endoplasmic reticulum of transfected cells. The cellular and molecular results in the two canine RPGR exon ORF15 mutations have implications for understanding the phenotypic variability found in human RP3 families that carry similar mutations.

  8. 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

  9. Implications of single nucleotide polymorphisms in CD44 exon 2 for risk of breast cancer.

    PubMed

    Zhou, Juhua; Nagarkatti, Prakash S; Zhong, Yin; Zhang, Jiajia; Nagarkatti, Mitzi

    2011-09-01

    CD44 is a cell-surface glycoprotein involved in many cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis and tumor metastasis, suggesting that CD44 may play an important role in breast cancer development. In this study, we examined whether CD44 exon 2 polymorphisms are associated with increased susceptibility to breast cancer. Direct nucleotide sequencing analysis showed that multiple single nucleotide polymorphisms were present in the CD44 exon 2 coding region in female patients with breast cancer. There was no significant difference in the frequency of any one single nucleotide polymorphism in the CD44 exon 2 coding region between patients with breast cancer and normal donors. However, CD44 polymorphisms in the CD44 exon 2 coding region were identified in approximately 40% of patients with breast cancer, which was significantly higher than in normal donors (odds ratio, 9.34; 95% confidence interval = 2.58-33.82; P < 0.0001). The Wilcoxon-Mann-Whitney test analysis showed that the patients with the CD44 polymorphisms in CD44 exon 2 coding sequence had breast cancer at earlier ages, 49 ± 3 versus 62 ± 2 years (P < 0.0005), and larger tumor burdens (4.9 ± 1.22 vs. 1.6 ± 0.15 mm, P < 0.01) at the time of diagnosis. Interestingly, African-American female patients having the CD44 polymorphisms in CD44 exon 2 coding sequence were diagnosed with breast cancer at very young age (41 ± 2 years). Our results show that CD44 exon 2 polymorphisms are associated with breast cancer development, and such analysis may be effectively used in the evaluation of risk, prediction of cancer, prevention, diagnosis, and epidemiological studies of breast cancer.

  10. Changes in alternative splicing of human and mouse genes are accompanied by faster evolution of constitutive exons.

    PubMed

    Cusack, Brian P; Wolfe, Kenneth H

    2005-11-01

    Alternative splicing is known to be an important source of protein sequence variation, but its evolutionary impact has not been explored in detail. Studying alternative splicing requires extensive sampling of the transcriptome, but new data sets based on expressed sequence tags aligned to chromosomes make it possible to study alternative splicing on a genome-wide scale. Although genes showing alternative splicing by exon skipping are conserved as compared to the genome as a whole, we find that genes where structural differences between human and mouse result in genome-specific alternatively spliced exons in one species show almost 60% greater nonsynonymous divergence in constitutive exons than genes where exon skipping is conserved. This effect is also seen for genes showing species-specific patterns of alternative splicing where gene structure is conserved. Our observations are not attributable to an inherent difference in rate of evolution between these two sets of proteins or to differences with respect to predictors of evolutionary rate such as expression level, tissue specificity, or genetic redundancy. Where genome-specific alternatively spliced exons are seen in mammals, the vast majority of skipped exons appear to be recent additions to gene structures. Furthermore, among genes with genome-specific alternatively spliced exons, the degree of nonsynonymous divergence in constitutive sequence is a function of the frequency of incorporation of these alternative exons into transcripts. These results suggest that alterations in alternative splicing pattern can have knock-on effects in terms of accelerated sequence evolution in constant regions of the protein.

  11. Differentially expressed three non-coding alternate exons at 5' UTR of regulatory type I beta subunit gene of mouse.

    PubMed

    Banday, Abdul Rouf; Azim, Shafquat; Tabish, Mohammad

    2012-04-01

    Prkar1b gene encodes regulatory type I, beta subunit (RIβ) of cAMP dependent protein kinase A in mouse. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian PKA, RIβ subunit is considered to be one of the important subunits for neuronal functions. This is involved in multiple forms of synaptic plasticity, and influences memory and learning by maintaining hippocampal long-term potentiation (LTP). Deficient expression of this gene has been implicated in autoimmune disease systemic lupus erythematosus (SLE). We have identified two novel non-coding exons of the Prkar1b gene (designated as exon 1A and exon 1B), which are spliced to the canonical exon 2 and constitute the 5' untranslated region giving rise to three alternative transcript isoforms. We have also confirmed the expression of the previously known first exon (designated as exon 1C) with known transcript published earlier. The transcripts containing exons 1A, 1B and 1C are differentially regulated during the development and tissue types. In silico study of more than 20 kb nucleotide sequence upstream of known translational initiation codon revealed three distinct promoter regions named as PA, PB, and PC upstream of the exon 1A, exon 1B and exon 1C respectively. PB is non-CpG related promoter but PA and PC are CpG related promoters, however all three promoters are TATA less. Further analysis showed that these promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Prkar1b gene.

  12. HEXEvent: a database of Human EXon splicing Events.

    PubMed

    Busch, Anke; Hertel, Klemens J

    2013-01-01

    HEXEvent (http://hexevent.mmg.uci.edu) is a new database that permits the user to compile genome-wide exon data sets of human internal exons showing selected splicing events. User queries can be customized based on the type and the frequency of alternative splicing events. For each splicing version of an exon, an ESTs count is given, specifying the frequency of the event. A user-specific definition of constitutive exons can be entered to designate an exon exclusion level still acceptable for an exon to be considered as constitutive. Similarly, the user has the option to define a maximum inclusion level for an exon to be called an alternatively spliced exon. Unlike other existing splicing databases, HEXEvent permits the user to easily extract alternative splicing information for individual, multiple or genome-wide human internal exons. Importantly, the generated data sets are downloadable for further analysis.

  13. Exon skipping therapy for Duchenne muscular dystrophy.

    PubMed

    Kole, Ryszard; Krieg, Arthur M

    2015-06-29

    Duchenne muscular dystrophy (DMD) is caused mostly by internal deletions in the gene for dystrophin, a protein essential for maintaining muscle cell membrane integrity. These deletions abrogate the reading frame and the lack of dystrophin results in progressive muscle deterioration. DMD patients experience progressive loss of ambulation, followed by a need for assisted ventilation, and eventual death in mid-twenties. By the method of exon skipping in dystrophin pre-mRNA the reading frame is restored and the internally deleted but functional dystrophin is produced. Two oligonucleotide drugs that induce desired exon skipping are currently in advanced clinical trials.

  14. Source Mechanisms and Stress Fields of the 15-16 June 2013 Crete Earthquake Sequence Along Hellenic Subduction Zone

    NASA Astrophysics Data System (ADS)

    Görgün, Ethem

    2017-03-01

    15 June 2013 M w 6.1 off-shore southern Crete earthquake and its aftershock sequence along Hellenic Subduction Zone are examined. Centroid moment tensors (CMTs) for 40 earthquakes with moment magnitudes ( M w) between 3.5 and 6.1 are determined by applying a waveform inversion method. The mainshock is shallow focus thrust event with a minor strike-slip component at a depth of 20 km. The seismic moment ( M o) of the mainshock is estimated as 2.07 × 1018 Nm, and rupture duration of the mainshock is 4 s. The focal mechanisms of aftershocks are mainly thrust faulting with a strike-slip component. The geometry of the moment tensors ( M w ≥ 3.5) reveals a thrust faulting regime with N-S trending direction of P axis in the entire activated region. According to high-resolution CMT solutions of the off-shore southern Crete earthquake sequence, one main cluster consisting of 40 events is revealed. The aftershock activity in the observation period between 15 June and 15 July 2013 extends from N to S and NW to SE directions. Seismic cross sections indicate a complex pattern of the hypocenter distribution with the activation of two segments. The subduction interface is clearly revealed with high-resolution hypocenter source relocation and moment tensor solution. The best-constrained focal depths indicate that the aftershock sequence is mainly confined in the upper plate (depth < 30 km) and is ranging from about 5-28 km depth. A stress tensor inversion of focal mechanism data is performed to obtain a more precise picture of the off-shore southern Crete stress field. The stress tensor inversion results indicate a predominant thrust stress regime with a NE-SW-oriented maximum horizontal compressive stress ( S H). According to variance of the stress tensor inversion, to first order, the southern Crete region is characterized by a homogeneous interplate stress field. We also investigate the Coulomb stress change associated with the mainshock to evaluate any significant

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

    PubMed Central

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

    2015-01-01

    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. PMID:26356813

  16. 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.

  17. 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.

  18. Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene.

    PubMed

    Mercado, Pablo Arrisi; Ayala, Youhna M; Romano, Maurizio; Buratti, Emanuele; Baralle, Francisco E

    2005-01-01

    Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)16 tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.

  19. 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.

  20. Limited HLA sequence variation outside of antigen recognition domain exons of 360 10 of 10 matched unrelated hematopoietic stem cell transplant donor-recipient pairs.

    PubMed

    Hou, L; Vierra-Green, C; Lazaro, A; Brady, C; Haagenson, M; Spellman, S; Hurley, C K

    2017-01-01

    Traditional DNA-based typing focuses primarily on interrogating the exons of human leukocyte antigen (HLA) genes that form the antigen recognition domain (ARD). The relevance of mismatching donor and recipient for HLA variation outside the ARD on hematopoietic stem cell transplantation (HSCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) matched unrelated donor transplant pairs (n = 360). Next-generation DNA sequencing was used to characterize both HLA exons and introns for HLA-A, -B, -C alleles; exons 2, 3 and the intervening intron for HLA-DRB1 and exons only for HLA-DQA1 and -DQB1. Over 97% of alleles at each locus were matched for their nucleotide sequence outside of the ARD exons. Of the 4320 allele comparisons overall, only 17 allele pairs were mismatched for non-ARD exons, 41 for noncoding regions and 9 for ARD exons. The observed variation between donor and recipient usually involved a single nucleotide difference (88% of mismatches); 88% of the non-ARD exon variants impacted the amino acid sequence. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D-R pairs will be difficult to assess in HSCT outcome studies because these mismatches do not occur very frequently.

  1. A survey on intron and exon lengths.

    PubMed Central

    Hawkins, J D

    1988-01-01

    The lengths of introns and exons in various parts of genes of vertebrates, insects, plants and fungi are tabulated. Differences between the various groups of organisms are apparent. The results are discussed and support the idea that, generally speaking, introns were present in primitive genomes, though in some cases they may have been inserted into pre-existing genes. PMID:3057449

  2. Homozygous deletion but not mutation of exons 5 and 8 of the fragile histidine triad (FHIT) gene is associated with features of differentiated thyroid carcinoma.

    PubMed

    Yin, De-Tao; Wang, Lin; Sun, Jianrei; Yin, Fengyan; Yan, Qingtao; Shen, Ru-Long; Gao, Jian-Xin; He, Gang

    2010-01-01

    The fragile histidine triad (FHIT) gene encompasses the most common human fragile site, FRA3B at 3p14.2, a region that is involved in homozygous deletions in a variety of human tumors. FHIT is considered to be a tumor suppressor gene that is frequently inactivated in various types of cancer. To study the role of the FHIT gene in thyroid tumorigenesis, we looked for homozygous deletions or mutations of exons 5 and 8 of the FHIT gene in 65 cases of differentiated thyroid carcinoma (DTC) and their matched non-cancerous epithelium (NCE), using exon-specific PCR amplification and PCR single strand conformation polymorphism (PCR-SSCP) techniques. In DTC, the incidence of homozygous deletion of exon 5 was 30.8% (20/65), and it was associated with tumor metastasis to lymph nodes (p <0.05). The incidence of homozygous deletion of exon 8 was 29.2% (19/65), and it was associated with the tumor pathological grade, TNM stage, and lymph node metastasis (p <0.05). There was strong correlation between homozygous deletions of exon 5 and exon 8 (p <0.01). No point mutations were observed in either exon 5 or exon 8. These findings suggest that: (a) exons 5 and 8 of FHIT are key target regions of deletion, (b) homozygous deletions of exon 5 and exon 8 may be good biomarkers for the biological behavior of DTC, and (c) point mutation of these exons may not be involved in the inactivation of the FHIT gene in DTC.

  3. RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

    PubMed Central

    Ou, Alexander C.; Park, Jennie; Yu, Faye; Yu, Brian; Lee, Angela; Yang, Guang; Zhou, Anyu; Benz, Edward J.

    2012-01-01

    The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5′ splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5′ splice site, but not the engineered strong 5′ splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5′ splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5′ splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5′ splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA–U1 snRNP complex through interactions with U1C. PMID:22083953

  4. 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

  5. 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

  6. 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.

  7. Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes.

    PubMed

    Wang, Jinhua; Smith, Philip J; Krainer, Adrian R; Zhang, Michael Q

    2005-01-01

    Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.

  8. FGFR2 exon IIIa and IIIc mutations in Crouzon, Jackson-Weiss, and Pfeiffer syndromes: Evidence for missense changes, insertions, and a deletion due to alternative RNA splicing

    SciTech Connect

    Meyers, G.A.; Przylepa, K.A.; Scott, A.F.

    1996-03-01

    Fibroblast growth factor receptor 2 (FGFR2) mutations have been associated with the craniosynostotic conditions Crouzon, Jackson-Weiss, and Pfeiffer syndromes. Previously, mutations were described in the exons IIIa and IIIc, which form the extracellular, third immunoglobulin-like domain (IgM) and adjacent linker regions, both of which are normally involved in ligand binding. For all three conditions, mutations were found in exon IIIc. Only in Crouzon syndrome were mutations identified in exon IIIa. In this study, 39 cases with one of these three conditions were screened for exon IIIa or IIIc mutations. Eleven mutations are reported in 17 unrelated cases. Mutations in exon IIIa are identified for not only Crouzon but also Jackson-Weiss and Pfeiffer syndromes. Four mutations in either exon IIIa or exon IIIc reported only in Crouzon syndrome are present also in one of the other two syndromes. Two insertions, one in exon IIIa in a Crouzon syndrome patient and the other in exon IIIc in a Pfeiffer syndrome patient, were observed. The latter mutation has the same alternative RNA splicing effect as a reported synonymous mutation for Crouzon syndrome. A missense mutation was detected in one Pfeiffer syndrome family in which two members had craniosynostosis without limb anomalies. The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders. 16 refs., 3 figs., 1 tab.

  9. The "alternative" choice of constitutive exons throughout evolution.

    PubMed

    Lev-Maor, Galit; Goren, Amir; Sela, Noa; Kim, Eddo; Keren, Hadas; Doron-Faigenboim, Adi; Leibman-Barak, Shelly; Pupko, Tal; Ast, Gil

    2007-11-01

    Alternative cassette exons are known to originate from two processes-exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 5' splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs) that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicing.

  10. Skipping multiple exons of dystrophin transcripts using cocktail antisense oligonucleotides.

    PubMed

    Echigoya, Yusuke; Yokota, Toshifumi

    2014-02-01

    Duchenne muscular dystrophy (DMD) is one of the most common and lethal genetic disorders, with 20,000 children per year born with DMD globally. DMD is caused by mutations in the dystrophin (DMD) gene. Antisense-mediated exon skipping therapy is a promising therapeutic approach that uses short DNA-like molecules called antisense oligonucleotides (AOs) to skip over/splice out the mutated part of the gene to produce a shortened but functional dystrophin protein. One major challenge has been its limited applicability. Multiple exon skipping has recently emerged as a potential solution. Indeed, many DMD patients need exon skipping of multiple exons in order to restore the reading frame, depending on how many base pairs the mutated exon(s) and adjacent exons have. Theoretically, multiple exon skipping could be used to treat approximately 90%, 80%, and 98% of DMD patients with deletion, duplication, and nonsense mutations, respectively. In addition, multiple exon skipping could be used to select deletions that optimize the functionality of the truncated dystrophin protein. The proof of concept of systemic multiple exon skipping using a cocktail of AOs has been demonstrated in dystrophic dog and mouse models. Remaining challenges include the insufficient efficacy of systemic treatment, especially for therapies that target the heart, and limited long-term safety data. Here we review recent preclinical developments in AO-mediated multiple exon skipping and discuss the remaining challenges.

  11. 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.

  12. Absence of the exon 1 coding sequence of the androgen receptor gene associated with teratozoospermia in a Brazilian population.

    PubMed

    Mesquita, W E J C; Approbato, M S; Moura, K K V O; Jesuíno, R S A

    2009-11-24

    The androgen receptor (AR) is a protein encoded by the AR gene, which when mutated may affect spermatogenesis, the process in which spermatozoa are produced; thus, AR mutations could lead to male infertility. We examined exon 1 of the AR gene in men with idiopathic infertility. Blood or semen samples from 111 infertile, oligozoospermic (N = 31), asthenozoospermic (N = 23), teratozoospermic (N = 33), and azoospermic (N = 24) men were analyzed. The extracted DNA was amplified for the exon 1 region of the AR gene. There was a significant correlation between the absence of exon 1 in the AR gene and spermatogenesis defects (P = 0.015). This association was significant in teratozoospermic men (51.5% of the sample). We found that lack of amplification of exon 1 of the AR gene by polymerase chain reaction is associated with morphological defects in the spermogram.

  13. Discussing epigenetics in Southern California: a report from the International Symposium on Epigenetic Control and Cellular Plasticity, UCI, December 15-16, 2011.

    PubMed

    Rattner, Barbara P

    2012-04-01

    With the goal of discussing how epigenetic control and chromatin remodeling contribute to the various processes that lead to cellular plasticity and disease, this symposium marks the collaboration between the Institut National de la Santé et de la Recherche Médicale (INSERM) in France and the University of California, Irvine (UCI). Organized by Paolo Sassone-Corsi (UCI) and held at the Beckman Center of the National Academy of Sciences at the UCI campus December 15-16, 2011, this was the first of a series of international conferences on epigenetics dedicated to the scientific community in Southern California. The meeting also served as the official kick off for the newly formed Center for Epigenetics and Metabolism at the School of Medicine, UCI (http://cem.igb.uci.edu).

  14. MicroRNA-10b inhibition reduces E2F1-mediated transcription and miR-15/16 activity in glioblastoma

    PubMed Central

    Teplyuk, Nadiya M.; Uhlmann, Erik J.; Wong, Andus Hon-Kit; Karmali, Priya; Basu, Meenakshi; Gabriely, Galina; Jain, Anant; Wang, Yang; Chiocca, E. Antonio; Stephens, Robert; Marcusson, Eric; Yi, Ming; Krichevsky, Anna M.

    2015-01-01

    MicroRNA-10b (miR-10b) is commonly elevated in glioblastoma (GBM), while not expressed in normal brain tissues. Targeted inhibition of miR-10b has pleiotropic effects on GBM derived cell lines, it reduces GBM growth in animal models, but does not affect normal neurons and astrocytes. This data raises the possibility of developing miR-10b-targeting GBM therapy. However, the mechanisms contributing to miR-10b-mediated glioma cell survival and proliferation are unexplored. We found that inhibition of miR-10b has distinct effects on specific glioma cell lines. In cells expressing high levels of tumor suppressor p21WAF1/Cip1, it represses E2F1-mediated transcription, leading to down-regulation of multiple E2F1 target genes encoding for S-phase specific proteins, epigenetic modulators, and miRNAs (e.g. miR-15/16), and thereby stalling progression through the S-phase of cell cycle. Subsequently, miR-15/16 activities are reduced and many of their direct targets are de-repressed, including ubiquitin ligase FBXW7 that destabilizes Cyclin E. Conversely, GBM cells expressing low p21 level, or after p21 knock-down, exhibit weaker or no E2F1 response to miR-10b inhibition. Comparative analysis of The Cancer Genome Atlas revealed a strong correlation between miR-10b and multiple E2F target genes in GBM and low-grade glioma. Taken together, these findings indicate that miR-10b regulates E2F1-mediated transcription in GBM, in a p21-dependent fashion. PMID:25738367

  15. 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

  16. EGFR exon 20 insertion mutation in Japanese lung cancer.

    PubMed

    Sasaki, Hidefumi; Endo, Katsuhiko; Takada, Minoru; Kawahara, Masaaki; Kitahara, Naoto; Tanaka, Hisaichi; Okumura, Meinoshin; Matsumura, Akihide; Iuchi, Keiji; Kawaguchi, Tomoya; Kawano, Osamu; Yukiue, Haruhiro; Yokoyama, Tomoki; Yano, Motoki; Fujii, Yoshitaka

    2007-12-01

    Mutations of the epidermal growth factor receptor (EGFR) gene have been reported in non-small cell lung cancer (NSCLC), especially in female, never smoker patients with adenocarcinoma. Some common somatic mutations in EGFR, including deletion mutations in exon 19 and leucine to arginine substitution at amino acid position 858 (L858R) in exon 21, have been examined for their ability to predict sensitivity to gefitinib or erlotinib. On the other hand, previous report has shown that the insertion mutation at exon 20 is related to gefitinib resistance. We investigated the exon 20 EGFR mutation statuses in 322 surgically treated non-small cell lung cancer cases. Two hundred and five adenocarcinoma cases were included. The presence or absence of EGFR mutations of kinase domains was analyzed by direct sequences. EGFR insertion mutations at exon 20 were found from 7 of 322 (2.17%) lung cancer patients. We also detected the 18 deletion type mutations in exon 19, and 25 L858R type mutations in exon 21. There was a tendency towards higher exon 20 insertion ratio in never smoker (never smoker 4.4% versus smoker 1.3%, p=0.0996) and female (female 4.5% versus male 1.3%, p=0.0917). Two exon 20 insertion cases were treated with gefitinib and failed to response. EGFR insertion mutation in exon 20 could not be ignored from Japanese lung cancers.

  17. MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3

    PubMed Central

    Gooding, Clare; Edge, Christopher; Lorenz, Mike; Coelho, Miguel B.; Winters, Mikael; Kaminski, Clemens F.; Cherny, Dmitry; Eperon, Ian C.; Smith, Christopher W.J.

    2013-01-01

    Exon 3 of the rat α-tropomyosin (Tpm1) gene is repressed in smooth muscle cells, allowing inclusion of the mutually exclusive partner exon 2. Two key types of elements affect repression of exon 3 splicing: binding sites for polypyrimidine tract-binding protein (PTB) and additional negative regulatory elements consisting of clusters of UGC or CUG motifs. Here, we show that the UGC clusters are bound by muscleblind-like proteins (MBNL), which act as repressors of Tpm1 exon 3. We show that the N-terminal region of MBNL1, containing its four CCCH zinc-finger domains, is sufficient to mediate repression. The same region of MBNL1 can make a direct protein-to-protein interaction with PTB, and RNA binding by MBNL promotes this interaction, apparently by inducing a conformational change in MBNL. Moreover, single molecule analysis showed that MBNL-binding sites increase the binding of PTB to its own sites. Our data suggest that the smooth muscle splicing of Tpm1 is mediated by allosteric assembly of an RNA–protein complex minimally comprising PTB, MBNL and their cognate RNA-binding sites. PMID:23511971

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

    PubMed

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

    2016-10-12

    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.

  20. 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.

  1. Gene identification using exon amplification on human chromosome 18q21: implications for bipolar disorder.

    PubMed

    Chen, H; Huo, Y; Patel, S; Zhu, X; Swift-Scanlan, T; Reeves, R H; DePaulo, R; Ross, C A; McInnis, M G

    2000-09-01

    We previously reported linkage between bipolar disorder and a region on human chromosome (HC) 18q21. To identify genes in this region, exon trapping was performed on cosmids isolated from an HC18-specific cosmid library (LL18NC02) using 47 sequence tagged site (STS) markers from 18q21 as hybridization probes. A total of 285 unique sequences (exons) were obtained from 850 sequenced clones. Homology searching of the databases using NCBI's BLAST algorithms revealed that 31 exons have identity to known genes and/or ESTs, seven are identical to regions of finished genomic sequences in the 18q21 region, 20 have significant similarity (>30% sequence identity) to genes from human and/or other species, 19 were repetitive sequences, and 208 sequences (72%) are novel. Seventy per cent of the trapped sequences were predicted to be derived from genes using library screening and RT-PCR analyses. This represents an initial stage in characterizing genes in a susceptibility region for further study in bipolar disorder or other diseases that map to this region.

  2. Methylation pattern of H19 exon 1 is closely related to preeclampsia and trophoblast abnormalities.

    PubMed

    Lu, Linshan; Hou, Zheng; Li, Li; Yang, Yanhong; Wang, Xiaohong; Zhang, Beilei; Ren, Mo; Zhao, Dan; Miao, Zhuo; Yu, Lili; Yao, Yuanqing

    2014-09-01

    Preeclampsia (PE) is a pregnancy-induced disorder characterized by the overproliferation of trophoblasts. Hydatidiform moles, which are associated with a high risk of developing PE, are characterized by the excessive proliferation of trophoblastic tissue. H19 is highly expressed in placental tissue; however, its biological function remains unclear. A fundamental modification of the H19 gene is DNA methylation, which typically occurs in CG-rich regions at the promoter or the first exon region. In this study, in order to investigate the DNA methylation pattern of the H19 exon 1 region in placental tissues and trophoblast cells, placental specimens were collected from women in the first trimester of pregrancy (FTP) and the third trimester of pregnancy (TTP), as well as from from women with severe preeclampsia (sPE). We found that the DNA methylation levels of H19 exon 1 were significantly higher in the tissues obtained from women in TTP than from those obtained from women in FFP. The methylation status of CpG 1 sites within exon 1 of H19 was markedly higher in the placental tissues obtained from women with sPE than in the tissues obtained from women in TTP. In addition, we used the human choriocarcinoma cell line, JEG-3, and treated the cells with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza‑Dc). Following treatment with 5-Aza-Dc, the methylation levels at this CpG site showed marked hypomethylation. In addtion, the cell proliferative, migratory and invasive capacities of the cells were remarkably inhibited. Our data suggest that hypermethylation at individual CpG sites within exon 1 of H19 may be involved in the dysfunction of trophoblasts and the pathogenesis of PE.

  3. 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.

  4. 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.

  5. 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.

  6. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: molecular pathology of mutations in PAH exon 11.

    PubMed

    Heintz, Caroline; Dobrowolski, Steven F; Andersen, Henriette Skovgaard; Demirkol, Mübeccel; Blau, Nenad; Andresen, Brage Storstein

    2012-08-01

    In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response.

  7. 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.

  8. 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.

  9. 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

  10. The many faces of rare diseases (RD): meeting report on Rare Disease in South-Eastern Europe, 15-16 November 2013, Skopje, Republic of Macedonia.

    PubMed

    Gucev, Z; Tasic, V; Polenakovic, M

    2014-03-01

    The Second meeting on Rare Diseases in South Eastern Europe (SEE) was held in Skope, Macedonia on November 15-16, 2013. Objective and main data: Rare diseases (RD) are a major problem in developed and especially in countries without affluence. 6-8% of every population suffers from RD. The cumulative effect of RDs on the health system of a country is increasing. Diagnosis often remains a challenge and requires international collaboration. Treatment in diseases for which medication exist is often inaccessible to patients because of the high costs. All countries of SEE need screening programs that address more diseases. Patient organizations play a major role in increasing awareness and providing the needed pressure on society to treat treatable RDs. On the other hand, RDs are frequently a source of valuable new molecular insights not only on mechanisms of their etiology and pathology, but sometimes provide an insight on mechanisms of frequent diseases in man. Further efforts are needed in improving all the RD aspects mentioned.

  11. 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.

  12. The deletion of exon 3 in the cardiac ryanodine receptor is rescued by β strand switching.

    PubMed

    Lobo, Paolo A; Kimlicka, Lynn; Tung, Ching-Chieh; Van Petegem, Filip

    2011-06-08

    Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the β trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca²⁺ release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.

  13. 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

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

    USGS Publications Warehouse

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

    2016-01-01

    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.

  15. Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in.

    PubMed

    Popplewell, Linda; Koo, Taeyoung; Leclerc, Xavier; Duclert, Aymeric; Mamchaoui, Kamel; Gouble, Agnés; Mouly, Vincent; Voit, Thomas; Pâques, Frédéric; Cédrone, Frédéric; Isman, Olga; Yáñez-Muñoz, Rafael J; Dickson, George

    2013-07-01

    Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.

  16. Characterization of major histocompatibility complex (MHC) DRB exon 2 and DRA exon 3 fragments in a primary terrestrial rabies vector (Procyon lotor).

    PubMed

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

    2010-08-10

    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 (250 bp) 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.

  17. Alternative splicing regulation of APP exon 7 by RBFox proteins.

    PubMed

    Alam, Shafiul; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2014-12-01

    RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues.

  18. High correspondence between Affymetrix exon and standard expression arrays.

    PubMed

    Okoniewski, Michał J; Hey, Yvonne; Pepper, Stuart D; Miller, Crispin J

    2007-02-01

    Exon arrays aim to provide comprehensive gene expression data at the level of individual exons, similar to that provided on a per-gene basis by existing expression arrays. This report describes the performance of Affymetrix GeneChip Human Exon 1.0 ST array by using replicated RNA samples from two human cell lines, MCF7 and MCF10A, hybridized both to Exon 1.0 ST and to HG-U133 Plus2 arrays. Cross-comparison between array types requires an appropriate mapping to be found between individual probe sets. Three possible mappings were considered, reflecting different strategies for dealing with probe sets that target different parts of the same transcript. Irrespective of the mapping used, Exon 1.0 ST and HG-U133 Plus2 arrays show a high degree of correspondence. More than 80% of HG-U133 Plus2 probe sets may be mapped to the Exon chip, and fold changes are found well preserved for over 96% of those probe sets detected present. Since HG-U133 Plus2 arrays have already been extensively validated, these results lend a significant degree of confidence to exon arrays.

  19. Exclusion of exon 2 is a common mRNA splice variant of primate telomerase reverse transcriptases.

    PubMed

    Withers, Johanna B; Ashvetiya, Tamara; Beemon, Karen L

    2012-01-01

    Telomeric sequences are added by an enzyme called telomerase that is made of two components: a catalytic protein called telomerase reverse transcriptase (TERT) and an integral RNA template (TR). Telomerase expression is tightly regulated at each step of gene expression, including alternative splicing of TERT mRNA. While over a dozen different alternative splicing events have been reported for human TERT mRNA, these were all in the 3' half of the coding region. We were interested in examining splicing of the 5' half of hTERT mRNA, especially since exon 2 is unusually large (1.3 kb). Internal mammalian exons are usually short, typically only 50 to 300 nucleotides, and most long internal exons are alternatively processed. We used quantitative RT-PCR and high-throughput sequencing data to examine the variety and quantity of mRNA species generated from the hTERT locus. We determined that there are approximately 20-40 molecules of hTERT mRNA per cell in the A431 human cell line. In addition, we describe an abundant, alternatively-spliced mRNA variant that excludes TERT exon 2 and was seen in other primates. This variant causes a frameshift and results in translation termination in exon 3, generating a 12 kDa polypeptide.

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

    PubMed

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

    2017-01-01

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

  1. Mutation in exon 7 of PTCH deregulates SHH/PTCH/SMO signaling: possible linkage to WNT.

    PubMed

    Musani, Vesna; Gorry, Philippe; Basta-Juzbasic, Aleksandra; Stipic, Tonci; Miklic, Pavle; Levanat, Sonja

    2006-05-01

    The novel PTCH mutation and clinical manifestations within Gorlin syndrome family links PTCH haploinsufficiency and aberrant activation of the Wnt pathway. We report a family case with Gorlin syndrome, characterized by the usual phenotype features such as widespread basocellular tumors and craniofacial and bone malformations, but also including a less common appearance of craniopharyngioma. These clinical manifestations might be associated with a novel constitutional mutation of the PTCH gene, 1047insAGAA, which we found in exon 7. It changes the normal amino acid sequence leading to termination of the PTCH protein at exon 9. The analyzed tumors of the family show extensive loss of heterozygosity in the PTCH region, both basocellular and in particular craniopharyngioma, and in the latter a high expression of beta-catenin was detected. Our findings suggest involvement of the SHH/PTCH/SMO pathway in pathogenesis of the analyzed disorders, including its possible contribution to aberrant activation of the Wnt pathway in craniopharyngioma.

  2. X:Map: annotation and visualization of genome structure for Affymetrix exon array analysis

    PubMed Central

    Yates, Tim; Okoniewski, Michał J.; Miller, Crispin J.

    2008-01-01

    Affymetrix exon arrays aim to target every known and predicted exon in the human, mouse or rat genomes, and have reporters that extend beyond protein coding regions to other areas of the transcribed genome. This combination of increased coverage and precision is important because a substantial proportion of protein coding genes are predicted to be alternatively spliced, and because many non-coding genes are known also to be of biological significance. In order to fully exploit these arrays, it is necessary to associate each reporter on the array with the features of the genome it is targeting, and to relate these to gene and genome structure. X:Map is a genome annotation database that provides this information. Data can be browsed using a novel Google-maps based interface, and analysed and further visualized through an associated BioConductor package. The database can be found at http://xmap.picr.man.ac.uk. PMID:17932061

  3. Exonic Sp1 sites are required for neural-specific expression of the glycine receptor beta subunit gene.

    PubMed Central

    Tintrup, H; Fischer, M; Betz, H; Kuhse, J

    2001-01-01

    The gene encoding the beta subunit of the inhibitory glycine receptor (GlyR) is widely expressed throughout the mammalian central nervous system. To unravel the elements regulating its transcription, we isolated its 5' non-coding and upstream flanking regions from mouse. Sequence analysis revealed significant differences between the 5' region of the beta subunit gene and the corresponding regions of the homologous GlyR alpha subunit genes; it also identified a novel exon (exon 0) that encodes most of the 5'-untranslated portion of the GlyR beta mRNA. Primer extension experiments disclosed multiple transcriptional start sites. Transfection experiments with luciferase reporter gene constructs showed that sequences encompassing 1.58 kb of upstream flanking region and 180 bp of exon 0 displayed high promoter activity in two neuroblastoma cell lines but not in non-neural cells. Analysis of various deletion constructs showed that the 5' flanking region preceding the transcriptional start sites silences expression in non-neural cells but is not essential for general promoter activity. In contrast, the deletion of sequences within exon 0 drastically decreased or abolished transcription; the removal of sequences harbouring Sp1 consensus sequences within exon 0 decreased expression specifically in a neuroblastoma cell line. Band-shift assays confirmed the binding of Sp1 to sites within the deleted sequence. Our results indicate that neural-specific expression of the GlyR beta subunit gene might depend on a direct interaction of Sp1 transcription factors with cis elements located downstream from transcription initiation sites. PMID:11256962

  4. Human glucose phosphate isomerase: Exon mapping and gene structure

    SciTech Connect

    Xu, Weiming; Lee, Pauline; Beutler, E.

    1995-10-10

    The structure of the gene for human glucose phosphate isomerase (GPI) has been determined. Three GPI clones were isolated from a human genomic library by using a full-length GPI cDNA probe and were characterized. Oligonucleotides based on the known cDNA sequence were used as primers in amplification and sequence analyses. This led to the identification of the exon-intron junctions. By this approach, 18 exons and 17 introns have been identified. The exons range in size from 44 to 431 nucleotides. The intronic sequences surrounding the exons provide useful information for the identification of mutations that give rise to human GPI deficiency associated with chronic hemolytic anemia. 13 refs., 4 figs., 1 tab.

  5. Systematic identification and analysis of exonic splicing silencers.

    PubMed

    Wang, Zefeng; Rolish, Michael E; Yeo, Gene; Tung, Vivian; Mawson, Matthew; Burge, Christopher B

    2004-12-17

    Exonic splicing silencers (ESSs) are cis-regulatory elements that inhibit the use of adjacent splice sites, often contributing to alternative splicing (AS). To systematically identify ESSs, an in vivo splicing reporter system was developed to screen a library of random decanucleotides. The screen yielded 141 ESS decamers, 133 of which were unique. The silencer activity of over a dozen of these sequences was also confirmed in a heterologous exon/intron context and in a second cell type. Of the unique ESS decamers, most could be clustered into groups to yield seven putative ESS motifs, some resembling known motifs bound by hnRNPs H and A1. Potential roles of ESSs in constitutive splicing were explored using an algorithm, ExonScan, which simulates splicing based on known or putative splicing-related motifs. ExonScan and related bioinformatic analyses suggest that these ESS motifs play important roles in suppression of pseudoexons, in splice site definition, and in AS.

  6. Disentangling Sources of Selection on Exonic Transcriptional Enhancers

    PubMed Central

    Agoglia, Rachel M.; Fraser, Hunter B.

    2016-01-01

    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. PMID:26500252

  7. Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

    PubMed Central

    Calì, Francesco; Ruggeri, Giuseppa; Vinci, Mirella; Meli, Concetta; Carducci, Carla; Leuzzi, Vincenzo; Pozzessere, Simone; Schinocca, Pietro; Ragalmuto, Alda; Chiavetta, Valeria; Miccichè, Salvatore

    2010-01-01

    A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics. PMID:19946181

  8. 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

  9. 5'- and 3'-terminal nucleotides in the FGFR2 ISAR splicing element core have overlapping roles in exon IIIb activation and exon IIIc repression.

    PubMed

    Jones, R B; Carstens, R P; Luo, Y; McKeehan, W L

    2001-09-01

    The cell type-specific, mutually-exclusive alternative splicing of the fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is tightly regulated. A sequence termed ISAR (intronic splicing activator and repressor) has been implicated as an important cis regulatory element in both activation of exon IIIb and repression of exon IIIc splicing in epithelial cells. In order to better understand how this single sequence could have dual roles, we transfected minigenes containing a series of 2-bp mutations in the 18 3'-most nucleotides of ISAR that we refer to as the ISAR core. Transfection of cells with dual-exon (IIIb and IIIc) minigenes revealed that mutation of terminal sequences of the core led to decreased exon IIIb inclusion and increased exon IIIc inclusion. Transfection of cells with single-exon IIIb minigenes and single-exon IIIc minigenes revealed that mutation of terminal sequences of the ISAR core led to decreased exon IIIb inclusion and increased exon IIIc inclusion, respectively. Nucleotides of the ISAR core responsible for exon IIIb activation appear to overlap very closely with those required for exon IIIc repression. We describe a model in which ISAR and a 5' intronic sequence known as IAS2 form a stem structure required for simultaneous exon IIIb activation and exon IIIc repression.

  10. Representation of DNA sequences in genetic codon context with applications in exon and intron prediction.

    PubMed

    Yin, Changchuan

    2015-04-01

    To apply digital signal processing (DSP) methods to analyze DNA sequences, the sequences first must be specially mapped into numerical sequences. Thus, effective numerical mappings of DNA sequences play key roles in the effectiveness of DSP-based methods such as exon prediction. Despite numerous mappings of symbolic DNA sequences to numerical series, the existing mapping methods do not include the genetic coding features of DNA sequences. We present a novel numerical representation of DNA sequences using genetic codon context (GCC) in which the numerical values are optimized by simulation annealing to maximize the 3-periodicity signal to noise ratio (SNR). The optimized GCC representation is then applied in exon and intron prediction by Short-Time Fourier Transform (STFT) approach. The results show the GCC method enhances the SNR values of exon sequences and thus increases the accuracy of predicting protein coding regions in genomes compared with the commonly used 4D binary representation. In addition, this study offers a novel way to reveal specific features of DNA sequences by optimizing numerical mappings of symbolic DNA sequences.

  11. Nr4a3, a possibile oncogenic factor for neuroblastoma associated with CpGi methylation within the third exon.

    PubMed

    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

    2014-05-01

    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.

  12. An RRM-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion.

    PubMed

    Collins, Katherine M; Kainov, Yaroslav A; Christodolou, Evangelos; Ray, Debashish; Morris, Quaid; Hughes, Timothy; Taylor, Ian A; Makeyev, Eugene V; Ramos, Andres

    2017-04-04

    RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3΄ intronic sites only. Here, we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1-ZnF unit recognizes a GGA-centered motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognizes a C-rich sequence, which explains its known interaction with the intronic 3΄ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10's broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping.

  13. 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.

  14. Alternatively spliced products lacking exon 12 dominate the expression of fragile X mental retardation 1 gene in human tissues.

    PubMed

    Fu, Xianguo; Zheng, Dezhu; Liao, Juan; Li, Qingqin; Lin, Yuxiang; Zhang, Duo; Yan, Aizhen; Lan, Fenghua

    2015-08-01

    Fragile X mental retardation 1 gene (FMR1) expression is associated with fragile X syndrome (FXS) and exhibits several splicing products. However, the proportion of spliced isoforms that are expressed in different tissues remains unclear. In the present study, long-chain reverse transcription-polymerase chain reaction with a T cloning-sequencing method was conducted in order to analyze the entire coding region of the FMR1 gene in human tissues. In particular, FXS-associated tissues were analyzed, including the brain and testis. Twenty alternatively spliced isoforms were observed among 271 recombinants, including six novel ones. The isoform that consisted of the entire FMR1 coding region (ISO1) accounted for a small proportion of all isoforms. Isoforms lacking exon 12 were the most abundant. In particular, spliced isoforms ISO7 and ISO17 were the most abundant. However, their relative abundance varied between the peripheral blood cells, and the testis and brain tissues. Bioinformatic analyses suggested that exon 12 may be the sole exon undergoing positive selection. The results of the present study suggested that the mechanisms underlying alternative splicing (AS) of the FMR1 gene may be more complex. Furthermore, the functions of alternatively spliced products lacking exon 12 require further investigation. The results of the present study provide novel insights into the association between AS and the structure and function of the FMR1 gene.

  15. A mutation in an alternative untranslated exon of hexokinase 1 associated with Hereditary Motor and Sensory Neuropathy – Russe (HMSNR)

    PubMed Central

    Hantke, Janina; Chandler, David; King, Rosalind; Wanders, Ronald JA; Angelicheva, Dora; Tournev, Ivailo; McNamara, Elyshia; Kwa, Marcel; Guergueltcheva, Velina; Kaneva, Radka; Baas, Frank; Kalaydjieva, Luba

    2009-01-01

    Hereditary Motor and Sensory Neuropathy – Russe (HMSNR) is a severe autosomal recessive disorder, identified in the Gypsy population. Our previous studies mapped the gene to 10q22-q23 and refined the gene region to ∼70 kb. Here we report the comprehensive sequencing analysis and fine mapping of this region, reducing it to ∼26 kb of fully characterised sequence spanning the upstream exons of Hexokinase 1 (HK1). We identified two sequence variants in complete linkage disequilibrium, a G>C in a novel alternative untranslated exon (AltT2) and a G>A in the adjacent intron, segregating with the disease in affected families and present in the heterozygote state in only 5/790 population controls. Sequence conservation of the AltT2 exon in 16 species with invariable preservation of the G allele at the mutated site, strongly favour the exonic change as the pathogenic mutation. Analysis of the Hk1 upstream region in mouse mRNA from testis and neural tissues showed an abundance of AltT2-containing transcripts generated by extensive, developmentally regulated alternative splicing. Expression is very low compared with ubiquitous Hk1 and all transcripts skip exon1, which encodes the protein domain responsible for binding to the outer mitochondrial membrane, and regulation of energy production and apoptosis. Hexokinase activity measurement and immunohistochemistry of the peripheral nerve showed no difference between patients and controls. The mutational mechanism and functional effects remain unknown and could involve disrupted translational regulation leading to increased anti-apoptotic activity (suggested by the profuse regenerative activity in affected nerves), or impairment of an unknown HK1 function in the peripheral nervous system (PNS). PMID:19536174

  16. Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5' exons and possible mechanism for the genesis of the 3' end of v-src.

    PubMed Central

    Dorai, T; Levy, J B; Kang, L; Brugge, J S; Wang, L H

    1991-01-01

    To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain c

  17. Sequencing of Candidate Genes in Dominican Families Implicates Both Rare Exonic and Common Non-Exonic Variants for Carotid Intima-Media Thickness at Bifurcation

    PubMed Central

    Wang, Liyong; Beecham, Ashley; Dueker, Nicole; Blanton, Susan H.; Rundek, Tatjana; Sacco, Ralph L.

    2015-01-01

    Background Through linkage and tagSNP-based association studies in 100 Dominican Republic (DR) families, we previously identified ANLN and AOAH (7p14.3) as candidate genes for carotid intima-media thickness at bifurcation (bIMT). Methods and Results Introns, exons and flanking regions of ANLN and AOAH were re-sequenced in 151 individuals from 9 families with evidence for linkage at 7p14.3. For common variants [CV, minor allele frequency (MAF) ≥ 5%], single variant-based analysis was performed. For rare variants (RV, MAF<5%), gene-based analysis aggregating all RVs within a gene was performed. CV analysis revealed the strongest signal at rs3815483 (P=0.0003) in ANLN and rs60023210 (P=0.00005) in AOAH. In ANLN, RV analysis found suggestive evidence for association with exonic RVs (P=0.08), and in particular non-synonymous RVs (P=0.04) but not with all RVs (P=0.15). The variant alleles of all non-synonymous RVs segregated with the major allele of rs3815483 and were associated with lower bIMT while a novel synonymous RV segregated with the minor allele of rs3815483 and was associated with greater bIMT. Additional analysis in 561 DR individuals found suggestive evidence for association with all ANLN non-synonymous RVs (P=0.08). In AOAH, no evidence for association with RVs was detected. Instead, conditional analysis revealed that multiple independent intronic CVs are associated with bIMT in addition to rs60023210. Conclusions We demonstrate the utility of using family-based studies to evaluate the contribution of RVs. Our data suggest two modes of genetic architecture underlying the linkage and association at ANLN (multiple exonic RVs) and AOAH (multiple intronic CVs with uncharacterized functions). PMID:26319989

  18. SMN2 exon 7 splicing is inhibited by binding of hnRNP A1 to a common ESS motif that spans the 3' splice site.

    PubMed

    Doktor, Thomas Koed; Schroeder, Lisbeth Dahl; Vested, Anne; Palmfeldt, Johan; Andersen, Henriette Skovgaard; Gregersen, Niels; Andresen, Brage Storstein

    2011-02-01

    Spinal Muscular Atrophy is caused by homozygous loss of SMN1 with phenotypic modulation by SMN2. SMN2 expresses only limited amounts of full-length transcript due to skipping of exon 7 caused by disruption of an SF2/ASF binding ESE. Additionally, hnRNP A1 has been reported to inhibit inclusion of SMN2 exon 7. We previously reported high similarity between the sequence spanning the 3' ss of SMN1 and SMN2 exon 7 and an hnRNP A1 binding ESS, which regulates MCAD exon 5 splicing. We show here that this 3' ss motif indeed functions as a crucial hnRNP A1 binding ESS, which inhibits inclusion of SMN1/2 exon 7 and is antagonized by the SMN1 ESE, but not by the inactive SMN2 sequence. Pull-down experiments revealed a specific interaction between hnRNP A1 and the 3' ss AG-dinucleotide, which could be disrupted by mutations shown to improve splicing in reporter minigenes. Genomic analyses revealed that in the human genome, 3' ss matching the SMN1/2 ESS motif region are much less abundant than 3' ss with a disrupted ESS motif. This indicates that this ESS may be a general splicing inhibitory motif, which binds hnRNP A1 and inhibits exon inclusion by binding to 3' ss harboring this ESS motif.

  19. Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation.

    PubMed

    Kominato, Yoshihiko; Ueki, Misuzu; Iida, Reiko; Kawai, Yasuyuki; Nakajima, Tamiko; Makita, Chikako; Itoi, Masako; Tajima, Yutaka; Kishi, Koichiro; Yasuda, Toshihiro

    2006-07-01

    Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site approximately 12 kb upstream of exon 1, which was previously believed to be the single transcription-starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription-starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.

  20. Antisense-mediated exon skipping to reframe transcripts.

    PubMed

    Turczynski, Sandrina; Titeux, Matthias; Pironon, Nathalie; Hovnanian, Alain

    2012-01-01

    Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.

  1. Exonic Re-Sequencing of the Chromosome 2q24.3 Parkinson's Disease Locus.

    PubMed

    Labbé, Catherine; Ogaki, Kotaro; Lorenzo-Betancor, Oswaldo; Carrasquillo, Minerva M; Heckman, Michael G; McCarthy, Allan; Soto-Ortolaza, Alexandra I; Walton, Ronald L; Lynch, Timothy; Siuda, Joanna; Opala, Grzegorz; Krygowska-Wajs, Anna; Barcikowska, Maria; Czyzewski, Krzysztof; Dickson, Dennis W; Uitti, Ryan J; Wszolek, Zbigniew K; Ross, Owen A

    2015-01-01

    Genome-wide association studies (GWAS) in Parkinson's disease (PD) have identified over 20 genomic regions associated with disease risk. Many of these loci include several candidate genes making it difficult to pinpoint the causal gene. The locus on chromosome 2q24.3 encompasses three genes: B3GALT1, STK39, and CERS6. In order to identify if the causal variants are simple missense changes, we sequenced all 31 exons of these three genes in 187 patients with PD. We identified 13 exonic variants including four non-synonymous and three insertion/deletion variants (indels). These non-synonymous variants and rs2102808, the GWAS tag SNP, were genotyped in three independent series consisting of a total of 1976 patients and 1596 controls. Our results show that the seven identified 2q24.3 coding variants are not independently responsible for the GWAS association signal at the locus; however, there is a haplotype, which contains both rs2102808 and a STK39 exon 1 6bp indel variant, that is significantly associated with PD risk (Odds Ratio [OR] = 1.35, 95% CI: 1.11-1.64, P = 0.003). This haplotype is more associated than each of the two variants independently (OR = 1.23, P = 0.005 and 1.10, P = 0.10, respectively). Our findings suggest that the risk variant is likely located in a non-coding region. Additional sequencing of the locus including promoter and regulatory regions will be needed to pinpoint the association at this locus that leads to an increased risk to PD.

  2. 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

  3. 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.

  4. 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

  5. How slow RNA polymerase II elongation favors alternative exon skipping.

    PubMed

    Dujardin, Gwendal; Lafaille, Celina; de la Mata, Manuel; Marasco, Luciano E; Muñoz, Manuel J; Le Jossic-Corcos, Catherine; Corcos, Laurent; Kornblihtt, Alberto R

    2014-05-22

    Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3' splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5' ss increases ETR-3 binding at the 3' ss and subsequent E9 skipping, whereas strengthening of the 5' ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5' ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.

  6. Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing

    PubMed Central

    Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini; Chui, Darryl; Wohlschlegel, James A; Black, Douglas L

    2016-01-01

    Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest. DOI: http://dx.doi.org/10.7554/eLife.19743.001 PMID:27882870

  7. Alternative splicing of micro-exons creates multiple forms of the insect cell adhesion molecule fasciclin I.

    PubMed

    McAllister, L; Rehm, E J; Goodman, G S; Zinn, K

    1992-03-01

    Fasciclin I is a homophilic cell adhesion molecule in insects that is dynamically expressed on a subset of axon pathways in the embryonic nervous system, and on a variety of other cells and tissues during development. The fasciclin I protein consists of four homologous 150 amino acid domains. In this article, we describe the complete sequence of the Drosophila fasciclin I (fasI) gene. The gene consists of 15 exons and is distributed over 14 kilobases of DNA. We examine the structure and temporal expression pattern of multiple fasciclin I mRNAs that differ in the lengths of their 3' untranslated regions. We also show that a highly conserved sequence at the end of the second domain can be altered by the addition of three or six amino acids that are encoded by two alternatively spliced 9 base pair (bp) micro-exons. In grasshopper fasciclin I mRNAs, there are 9 bp and 6 bp insertions at the same position. The first of these insertions is identical in sequence to the first fly micro-exon. The grasshopper insertions are not found together in the same mRNA, so grasshopper fasciclin I species differ by the addition of three or two extra amino acids to the second domain. The alternatively spliced mRNAs are differentially expressed during embryogenesis, and all three of them are present in nerve cord preparations. We suggest that the amino acids inserted by alternative micro-exon splicing may alter the binding specificity of fasciclin I.

  8. 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.

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

    PubMed

    Kim, Hyunwoo; Park, Heejin; Paek, Eunok

    2015-07-02

    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.

  10. 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.

  11. Gene duplication, exon gain and neofunctionalization of OEP16-related genes in land plants.

    PubMed

    Drea, Sinéad C; Lao, Nga T; Wolfe, Kenneth H; Kavanagh, Tony A

    2006-06-01

    OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16-related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein. We analysed the sequence, structure and expression of the corresponding Arabidopsis genes (atOEP16-S and atOEP16-L) and demonstrated that following duplication, both genes diverged in terms of expression patterns and coding sequence. AtOEP16-S, which contains multiple G-box ABA-responsive elements (ABREs) in the promoter region, is regulated by ABI3 and ABI5 and is strongly expressed during the maturation phase in seeds and pollen grains, both desiccation-tolerant tissues. In contrast, atOEP-L, which lacks promoter ABREs, is expressed predominantly in leaves, is induced strongly by low-temperature stress and shows weak induction in response to osmotic stress, salicylic acid and exogenous ABA. Our results indicate that gene duplication, exon gain and regulatory sequence evolution each played a role in the divergence of OEP16 homologues in plants.

  12. 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.

  13. EXONSAMPLER: a computer program for genome-wide and candidate gene exon sampling for targeted next-generation sequencing.

    PubMed

    Cosart, Ted; Beja-Pereira, Albano; Luikart, Gordon

    2014-11-01

    The computer program EXONSAMPLER automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next-generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User-adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of EXONSAMPLER to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon-capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16,000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.

  14. 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.

  15. Exon skipping creates novel splice variants of DMC1 gene in ruminants.

    PubMed

    Ahlawat, S; Chopra, M; Jaiswal, L; Sharma, R; Arora, R; Brahma, B; Lal, S V; De, S

    2016-04-01

    Disrupted meiotic cDNA1 (DMC1) recombinase plays a pivotal role in homology search and strand exchange reactions during meiotic homologous recombination. In the present study, full length coding sequence of DMC1 gene was sequence characterized for the first time from four ruminant species (cattle, buffalo, sheep and goat) and phylogenetic relationship of ruminant DMC1 with other eukaryotes was analyzed. DMC1 gene encodes a putative protein of 340 amino acids in cattle, sheep and buffalo and 341 amino acids in goat. A high degree of evolutionary conservation at both nucleotide and amino acid level was observed for the four ruminant orthologs. In cattle and sheep, novel alternatively spliced mRNAs with skipping of exons 7 and 8 (Transcript variant 1, TV1) were isolated in addition to the full length (FL) transcript. Novel transcript variants with partial skipping of exon 7 and complete skipping of exon 8 (Transcript variant 2, TV2) were found in sheep and goat. The presence of these variants was validated by amplifying cDNA isolated from testis tissue of ruminants using two oligonucleotides flanking the deleted region. To accurately estimate their relative proportions, real-time PCR was performed using primers specific for each variant. Expression level of DMC1-FL was significantly higher than that of TV1 in cattle and TV2 in goat (P < 0.05). Relative ratio for expression of DMC1-FL: TV1: TV2 in sheep was 6.78: 1.43: 1. In-silico analysis revealed presence of splice variants of DMC1 gene across other mammalian species underpinning the role of alternative splicing in functional innovation.

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

    PubMed

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

    2014-10-30

    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.

  17. Alternative exon skipping biases substrate preference of the deubiquitylase USP15 for mysterin/RNF213, the moyamoya disease susceptibility factor

    PubMed Central

    Kotani, Yuri; Morito, Daisuke; Sakata, Kenshiro; Ainuki, Shiori; Sugihara, Munechika; Hatta, Tomohisa; Iemura, Shun-ichiro; Takashima, Seiji; Natsume, Tohru; Nagata, Kazuhiro

    2017-01-01

    The deubiquitylating enzyme USP15 plays significant roles in multiple cellular pathways including TGF-β signaling, RNA splicing, and innate immunity. Evolutionarily conserved skipping of exon 7 occurs during transcription of the mRNAs encoding USP15 and its paralogue USP4, yielding two major isoforms for each gene. Exon 7 of USP15 encodes a serine-rich stretch of 29 amino acid residues located in the inter-region linker that connects the N-terminal putative regulatory region and the C-terminal enzymatic region. Previous findings suggested that the variation in the linker region leads to functional differences between the isoforms of the two deubiquitylating enzymes, but to date no direct evidence regarding such functional divergence has been published. We found that the long isoform of USP15 predominantly recognizes and deubiquitylates mysterin, a large ubiquitin ligase associated with the onset of moyamoya disease. This observation represents the first experimental evidence that the conserved exon skipping alters the substrate specificity of this class of deubiquitylating enzymes. In addition, we found that the interactomes of the short and long isoforms of USP15 only partially overlapped. Thus, USP15, a key gene in multiple cellular processes, generates two functionally different isoforms via evolutionarily conserved exon skipping. PMID:28276505

  18. 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

  19. Genotyping of Intron Inversions and Point Mutations in Exon 14 of the FVIII Gene in Iranian Azeri Turkish Families with Hemophilia A.

    PubMed

    Shekari Khaniani, Mahmoud; Ebrahimi, Abdollah; Daraei, Setareh; Derakhshan, Sima Mansoori

    2016-12-01

    Hemophilia A (HA) is an inherited X-linked bleeding disorder caused by a variety of mutations that are distributed throughout the large FVIII gene (F8). The most common mutations in studied populations with severe HA are introns 22 and 1 inversions, gross exon deletions and point mutations in exon 14. The aim of this study was to define the frequency of these common mutations in Iranian population of Azeri Turkish in North West of Iran. Fifty patients with severe HA and forty-three female potential carriers were genotyped by inverse shifting polymerase chain reaction (IS-PCR), long-range PCR, multiplex PCR, and sequencing methods for the detection of Intron 22 and 1 inversions, gross exon deletions, and exon 14 point mutations, respectively. F8 intron 22 inversion was detected in 22 (44 %) out of 50 patients. Moreover, we detected one intron 1 inversion (2 %), and one point mutation in exon 14 (2 %). In this population, 52 % of the patients with hemophilia A did not show to carry a mutation in the analyzed regions by three mentioned methods. F8 intron 22 inversion was the major causative mutation in nearly 50 % of severe HA cases in an Azerbaijani Turkish population, which is similar to the incidence of other populations. IS-PCR is a robust, rapid, efficient, and cost-effective method for the genetic analysis of patients with severe HA and for HA carrier detection, especially in developing countries.

  20. Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites.

    PubMed

    Shepard, Peter J; Choi, Eun-A; Busch, Anke; Hertel, Klemens J

    2011-11-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3' and 5' splice sites. However, it is unknown if the 3' or the 5' splice site dominates the exon recognition process. Here, we test the 3' and 5' splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3' and 5' splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3' or the 5' splice site strength alone.

  1. Efficient use of a translation start codon in BDNF exon I.

    PubMed

    Koppel, Indrek; Tuvikene, Jürgen; Lekk, Ingrid; Timmusk, Tõnis

    2015-09-01

    The brain-derived neurotrophic factor (BDNF) gene contains a number of 5' exons alternatively spliced with a common 3' exon. BDNF protein is synthesized from alternative transcripts as a prepro-precursor encoded by the common 3' exon IX, which has a translation start site 21 bp downstream of the splicing site. BDNF mRNAs containing exon I are an exception to this arrangement as the last three nucleotides of this exon constitute an in-frame AUG. Here, we show that this AUG is efficiently used for translation initiation in PC12 cells and cultured cortical neurons. Use of exon I-specific AUG produces higher levels of BDNF protein than use of the common translation start site, resulting from a higher translation rate. No differences in protein degradation, constitutive or regulated secretion were detected between BDNF isoforms with alternative 5' termini. As the BDNF promoter preceding exon I is known to be highly regulated by neuronal activity, our results suggest that the function of this translation start site may be efficient stimulus-dependent synthesis of BDNF protein. The brain-derived neurotrophic factor (BDNF) gene contains multiple untranslated 5' exons alternatively spliced to one common protein-coding 3' exon. However, exon I contains an in-frame ATG in a favorable translation context. Here, we show that use of this ATG is associated with more efficient protein synthesis than the commonly used ATG in exon IX.

  2. 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.

  3. 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.

  4. Multiple splicing defects in an intronic false exon.

    PubMed

    Sun, H; Chasin, L A

    2000-09-01

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

  5. A two-stage exon recognition model based on synergetic neural network.

    PubMed

    Huang, Zhehuang; Chen, Yidong

    2014-01-01

    Exon recognition is a fundamental task in bioinformatics to identify the exons of DNA sequence. Currently, exon recognition algorithms based on digital signal processing techniques have been widely used. Unfortunately, these methods require many calculations, resulting in low recognition efficiency. In order to overcome this limitation, a two-stage exon recognition model is proposed and implemented in this paper. There are three main works. Firstly, we use synergetic neural network to rapidly determine initial exon intervals. Secondly, adaptive sliding window is used to accurately discriminate the final exon intervals. Finally, parameter optimization based on artificial fish swarm algorithm is used to determine different species thresholds and corresponding adjustment parameters of adaptive windows. Experimental results show that the proposed model has better performance for exon recognition and provides a practical solution and a promising future for other recognition tasks.

  6. Use of an engineered ribozyme to produce a circular human exon.

    PubMed Central

    Mikheeva, S; Hakim-Zargar, M; Carlson, D; Jarrell, K

    1997-01-01

    We report the use of an engineered ribozyme to produce a circular human exon in vitro. Specifically, we have designed a derivative of a yeast self-splicing group II intron that is able to catalyze the formation of a circular exon encoding the first kringle domain (K1) of the human tissue plasminogen activator protein. We show that the circular K1 exon is formed with high fidelity in vitro. Furthermore, the system is designed such that the circular exon that is produced consists entirely of human exon sequence. Thus, our results demonstrate that all yeast exon sequences are dispensable for group II intron catalyzed inverse splicing. This is the first demonstration that an engineered ribozyme can be used to create a circular exon containing only human sequences, linked together at a precise desired ligation point. We expect these results to be generalizable, so that similar ribozymes can be designed to precisely create circular derivatives of any nucleotide sequence. PMID:9396820

  7. 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

  8. Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia

    PubMed Central

    Gray, Mary J.; Kannu, Peter; Sharma, Swarkar; Neyt, Christine; Zhang, Dongping; Paria, Nandina; Daniel, Philip B.; Whetstone, Heather; Sprenger, Hans-Georg; Hammerschmidt, Philipp; Weng, Angela; Dupuis, Lucie; Jobling, Rebekah; Mendoza-Londono, Roberto; Dray, Michael; Su, Peiqiang; Wilson, Megan J.; Kapur, Raj P.; McCarthy, Edward F.; Alman, Benjamin A.; Howard, Andrew; Somers, Gino R.; Marshall, Christian R.; Manners, Simon; Flanagan, Adrienne M.; Rathjen, Karl E.; Karol, Lori A.; Crawford, Haemish; Markie, David M.; Rios, Jonathan J.; Wise, Carol A.; Robertson, Stephen P.

    2015-01-01

    The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (METΔ14) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the METΔ14 mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone. PMID:26637977

  9. The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains.

    PubMed Central

    Ny, T; Elgh, F; Lund, B

    1984-01-01

    A genomic clone carrying the human tissue-type plasminogen activator (t-PA) gene was isolated from a cosmid library, and the gene structure was elucidated by restriction mapping, Southern blotting, and DNA sequencing. The cosmid contained all the coding parts of the mRNA, except for the first 58 bases in the 5' end of the mRNA, and had a total length of greater than 20 kilobases. It was separated into at least 14 exons by at least 13 introns, and the exons seemed to code for structural or functional domains. Thus, the signal peptide, the propeptide, and the domains of the heavy chain, including the regions homologous to growth factors, and to the "finger" structure of fibronectin, are all encoded by separate exons. In addition, the two kringle regions of t-PA were both coded for by two exons and were cleaved by introns at identical positions. The region coding for the light chain, comprising the serine protease part of the molecule was split by four introns, revealing a gene organization similar to other serine proteases. PMID:6089198

  10. 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

  11. 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

    2006-05-04

    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.

  12. 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

  13. Splicing repression allows the gradual emergence of new Alu-exons in primate evolution

    PubMed Central

    Attig, Jan; Ruiz de los Mozos, Igor; Haberman, Nejc; Wang, Zhen; Emmett, Warren; Zarnack, Kathi; König, Julian; Ule, Jernej

    2016-01-01

    Alu elements are retrotransposons that frequently form new exons during primate evolution. Here, we assess the interplay of splicing repression by hnRNPC and nonsense-mediated mRNA decay (NMD) in the quality control and evolution of new Alu-exons. We identify 3100 new Alu-exons and show that NMD more efficiently recognises transcripts with Alu-exons compared to other exons with premature termination codons. However, some Alu-exons escape NMD, especially when an adjacent intron is retained, highlighting the importance of concerted repression by splicing and NMD. We show that evolutionary progression of 3' splice sites is coupled with longer repressive uridine tracts. Once the 3' splice site at ancient Alu-exons reaches a stable phase, splicing repression by hnRNPC decreases, but the exons generally remain sensitive to NMD. We conclude that repressive motifs are strongest next to cryptic exons and that gradual weakening of these motifs contributes to the evolutionary emergence of new alternative exons. DOI: http://dx.doi.org/10.7554/eLife.19545.001 PMID:27861119

  14. Sensitive detection of deletions of one or more exons in the neurofibromatosis type 2 (NF2) gene by multiplexed gene dosage polymerase chain reaction.

    PubMed

    Diebold, Ruth; Bartelt-Kirbach, Britta; Evans, D Gareth; Kaufmann, Dieter; Hanemann, C Oliver

    2005-02-01

    Mutation detection in the neurofibromatosis type 2 (NF2) gene is challenging because when combining mutation detection methods such as single-strand conformational polymorphism and heteroduplex analysis, denaturing gradient gel electrophoresis, and direct sequencing of aberrant polymerase chain reaction (PCR) fragments only 30 to 60% of the constitutional mutations are detected. Because large deletions and complete chromosome rearrangements are also described methods such as microarray-comparative genomic hybridization and fluorescence in situ hybridization are also used. The one type of mutation often missed corresponds to deletions encompassing one or few exons. To detect this type we have developed a swift and reliable method. We perform a gene dosage analysis with two fluorescent multiplex PCR assays that amplify 15 of the 17 NF2 exons. The labeled PCR products are quantified and gene dose is calculated with respect to controls. We tested the reliability of this method with DNA from eight NF2 patients with known heterozygous NF2 deletions, eight controls and four unknown NF2 patients. In all of the patients with known heterozygous deletions we found in several exons a reduction of gene dosage to 50 to 69%. In one NF2 patient with previously unknown mutation and a severe phenotype we found the gene dosage of two exons reduced by 50% indicating a deletion of these two exons on one allele. This finding was validated by reverse transcriptase-PCR on fibroblast and schwannoma cell cultures of this patient and cDNA sequencing. Our gene dosage assay will detect deletions of one or more exons as well as gross deletions of the whole coding region of the gene. It can complement the existing screening methods because it is faster and easier.

  15. Sequencing of the coding exons of the LRP1 and LDLR genes on individual DNA samples reveals novel mutations in both genes.

    PubMed

    Van Leuven, F; Thiry, E; Lambrechts, M; Stas, L; Boon, T; Bruynseels, K; Muls, E; Descamps, O

    2001-02-15

    Five coding polymorphisms in de LRP1 gene, i.e. A217V, A775P, D2080N, D2632E and G4379S were discovered by sequencing its 89 exons in three test-groups of 22 healthy individuals, 29 Alzheimer patients and 18 individuals with different clinical and molecularly uncharacterized lipid metabolism problems. No genetic defect was evident in the LRP1 gene of any of the Alzheimer's disease (AD) patients, further excluding LRP1 as a major genetic problem in AD. Lipoprotein receptor related protein (LRP) A217V (exon 6) was clearly present in all groups as a polymorphism, while D2632E was observed only once in a healthy volunteer. On the other hand, LRP1 alleles A775P, D2080N, and G4379 were encountered only in patients with FH or with undefined problems of lipid metabolism. This finding forced one to also analyze the LDL receptor (LDLR) gene, for which a method was devised to sequence the entire region comprising LDLR exons 2-18. The resulting sequence contig of 33567 nucleotides yielded finally an exact physical map that corrects published and listed LDLR gene maps in many positions. In addition, next to known mutations in LDLR that cause FH, four novel LDLR defects were defined, i.e. del e7-10, exon 9 mutation N407T, a 20 bp insertion in exon 4, and a double mutation C292W/K290R in exon 6. No evidence for pathology connected to the LRP1 'mutations' was obtained by subsequent screening for the five LRP1 variants in larger groups of 110 FH patients and 118 patients with molecularly undefined, clinical problems of cholesterol and/or lipid metabolism. In three individuals with a mutant LDLR gene a variant LRP1 allele was also present, but without direct, obvious clinical compound effects, indicating that the variant LRP1 alleles must, for the present, be considered polymorphisms.

  16. 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.

  17. Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25.

    PubMed

    Runkel, Fabian; Büssow, Heinrich; Seburn, Kevin L; Cox, Gregory A; Ward, Diane McVey; Kaplan, Jerry; Franz, Thomas

    2006-03-01

    The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.

  18. Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing: potential clinical implications.

    PubMed

    Marchetti, Antonio; Del Grammastro, Maela; Filice, Giampaolo; Felicioni, Lara; Rossi, Giulio; Graziano, Paolo; Sartori, Giuliana; Leone, Alvaro; Malatesta, Sara; Iacono, Michele; Guetti, Luigi; Viola, Patrizia; Mucilli, Felice; Cuccurullo, Franco; Buttitta, Fiamma

    2012-01-01

    Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.

  19. WT1 exon 1 deletion/insertion mutations in Wilms tumor patients, associated with di- and trinucleotide repeats and deletion hotspot consensus sequences.

    PubMed Central

    Huff, V; Jaffe, N; Saunders, G F; Strong, L C; Villalba, F; Ruteshouser, E C

    1995-01-01

    The WT1 gene is known to play a role in at least some cases of Wilms tumor (WT). The first exon of the gene is highly GC rich and contains many short tandem di- and trinucleotide repeats, interrupted direct repeats, and CCTG (CAGG) motifs that have been identified as hotspots for DNA deletions. We have analyzed 80 WT patient samples for mutations in the first exon of WT1, either by SSCP analysis of the first 131 bp of the coding portion of WT1 exon 1 or by size analysis of a PCR product encompassing the coding region of exon 1 in addition to flanking noncoding regions. We report here the occurrence of somatic and germ-line deletion and insertion mutations in this portion of the gene in four WT patients. The mutations are flanked by short direct repeats, and the breakpoints are within 5 nt of a CCTG (CAGG) sequence. These data suggest that a distinctive mutational mechanism, previously unrecognized for this gene, is important for the generation of DNA mutations at the WT1 locus. Images Figure 2 Figure 3 Figure 4 PMID:7825606

  20. NMR structure of the 5' splice site in the group IIB intron Sc.ai5γ--conformational requirements for exon-intron recognition.

    PubMed

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

    2014-03-01

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

  1. Evidence that the SRY protein is encoded by a single exon on the human Y chromosome

    SciTech Connect

    Behlke, M.A. Women's Hospital and Harvard Medical School, Boston, MA ); Bogan, J.S.; Beer-Romero, P.; Page, D.C. )

    1993-09-01

    To facilitate studies of the SRY gene, a 4741-bp portion of the sex-determining region of the human Y chromosome was sequenced and characterized. Two RNAs were found to hybridize to this genomic segment, one transcript deriving from SRY and the second cross-hybridizing to a pseudogene located 2.5 kb 5[prime] of the SRY open reading frame (ORF). Analysis of the SRY transcript using 3[prime] and 5[prime] rapid amplification and cloning of ends suggested that the entire SRY protein is encoded by a single exon. A 700-bp CpG island is located immediately 5[prime] of the pseudogene (and 2 kb 5[prime] of the SRY ORF). Within this CpG island lies the sequence CGCCCCCGC, a potential binding site for the EGR-1/WT1 family of transcription factors, some of which appear to function in gonadal development. 19 refs., 1 fig.

  2. Characterization of six mutations in Exon 37 of neurofibromatosis type 1 gene

    SciTech Connect

    Upadhyaya, M.; Osborn, M.; Maynard, J.; Harper, P.

    1996-07-26

    Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders, with an incidence of 1 in 3,000. We screened a total of 320 unrelated NF1 patients for mutations in exon 37 of the NF1 gene. Six independent mutations were identified, of which three are novel, and these include a recurrent nonsense mutation identified in 2 unrelated patients at codon 2281 (G2281X), a 1-bp insertion (6791 ins A) resulting in a change of TAG (tyrosine) to a TAA (stop codon), and a 3-bp deletion (6839 del TAC) which generated a frameshift. Another recurrent nonsense mutation, Y2264X, which was detected in 2 unrelated patients in this study, was also previously reported in 2 NF1 individuals. All the mutations were identified within a contiguous 49-bp sequence. Further studies are warranted to support the notion that this region of the gene contains highly mutable sequences. 17 refs., 2 figs., 1 tab.

  3. Detection of a novel mutation in exon 20 of the BRCA1 gene.

    PubMed

    Chakraborty, Abhijit; Katarkar, Atul; Chaudhuri, Keya; Mukhopadhyay, Ashis; Basak, Jayasri

    2013-12-01

    Hereditary breast cancer constitutes 5-10% of all breast cancer cases. Inherited mutations in the BRCA1 and BRCA2 tumor-suppressor genes account for the majority of hereditary breast cancer cases. The BRCA1 C-terminal region (BRCT) has a functional duplicated globular domain, which helps with DNA damage repair and cell cycle checkpoint protein control. More than 100 distinct BRCA1 missense variants with structural and functional effects have been documented within the BRCT domain. Interpreting the results of mutation screening of tumor-suppressor genes that can have high-risk susceptibility mutations is increasingly important in clinical practice. This study includes a novel mutation, p.His1746 Pro (c.5237A>C), which was found in BRCA1 exon 20 of a breast cancer patient. In silico analysis suggests that this mutation could alter the stability and orientation of the BRCT domain and the differential binding of the BACH1 substrate.

  4. 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.

  5. 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

  6. 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.

  7. A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons

    PubMed Central

    Han, Jungwon; Lee, Jong-Hyuk; Park, Sunyoung; Yoon, Soomin; Yoon, Aerin; Hwang, Do B; Lee, Hwa K; Kim, Min S; Lee, Yujean; Yang, Won J; Youn, Hong-Duk; Kim, Hyori; Chung, Junho

    2016-01-01

    The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition. PMID:27857068

  8. Testing the exon theory of genes: the evidence from protein structure.

    PubMed

    Stoltzfus, A; Spencer, D F; Zuker, M; Logsdon, J M; Doolittle, W F

    1994-07-08

    A tendency for exons to correspond to discrete units of protein structure in protein-coding genes of ancient origin would provide clear evidence in favor of the exon theory of genes, which proposes that split genes arose not by insertion of introns into unsplit genes, but from combinations of primordial mini-genes (exons) separated by spacers (introns). Although putative examples of such correspondence have strongly influenced previous debate on the origin of introns, a general correspondence has not been rigorously proved. Objective methods for detecting correspondences were developed and applied to four examples that have been cited previously as evidence of the exon theory of genes. No significant correspondence between exons and units of protein structure was detected, suggesting that the putative correspondence does not exist and that the exon theory of genes is untenable.

  9. Personalized exon skipping strategies to address clustered non-deletion dystrophin mutations.

    PubMed

    Forrest, Sarah; Meloni, Penny L; Muntoni, Francesco; Kim, Jihee; Fletcher, Sue; Wilton, Steve D

    2010-12-01

    Antisense oligomer induced exon skipping is showing promise as a therapy to reduce the severity of Duchenne muscular dystrophy. To date, the focus has been on excluding single exons flanking frame-shifting deletions in the dystrophin gene. However, a third of all Duchenne muscular dystrophy causing mutations are more subtle DNA changes. Thirty nine dystrophin exons are potentially frame-shifting and mutations in these will require the targeted removal of exon blocks to generate in-frame transcripts. We report that clustered non-deletion mutations in the dystrophin gene respond differently to different antisense oligomer preparations targeting the same dual exon block, the removal of which bypasses the mutation and restores the open reading-frame. The personalized nature of the responses to antisense oligomer application presents additional challenges to the induction of multi-exon skipping with a single oligomer preparation.

  10. Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers.

    PubMed

    Goren, Amir; Ram, Oren; Amit, Maayan; Keren, Hadas; Lev-Maor, Galit; Vig, Ida; Pupko, Tal; Ast, Gil

    2006-06-23

    Exonic splicing regulatory sequences (ESRs) are cis-acting factor binding sites that regulate constitutive and alternative splicing. A computational method based on the conservation level of wobble positions and the overabundance of sequence motifs between 46,103 human and mouse orthologous exons was developed, identifying 285 putative ESRs. Alternatively spliced exons that are either short in length or contain weak splice sites show the highest conservation level of those ESRs, especially toward the edges of exons. ESRs that are abundant in those subgroups show a different distribution between constitutively and alternatively spliced exons. Representatives of these ESRs and two SR protein binding sites were shown, experimentally, to display variable regulatory effects on alternative splicing, depending on their relative locations in the exon. This finding signifies the delicate positional effect of ESRs on alternative splicing regulation.

  11. 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

  12. Identification of AcMNPV EXON0 (ac141) domains required for efficient production of budded virus, dimerization and association with BV/ODV-C42 and FP25.

    PubMed

    Fang, Minggang; Nie, Yingchao; Dai, Xiaojiang; Theilmann, David A

    2008-05-25

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late gene exon0 (ac141) is required for the efficient production of budded virus (BV). EXON0 interacts with nucleopcapsid protein BV/ODV-C42 and FP25 and enables egress of nucleocapsids from the nucleus to the cytoplasm. This study examines the functional domains of EXON0 that play a role in BV production. Six putative domains of the 261 amino acid EXON0 were deleted and examined for functionality by determining their ability to rescue an AcMNPV exon0 knockout bacmid. Domain mapping results showed that all the six domains were required but deletion of the N-terminal acidic region and the leucine zipper domains had the greatest impact on BV production. Yeast 2-hybrid and co-immunoprecipitation demonstrated that EXON0 formed dimers. Point mutation analysis demonstrated that the leucine zipper was required for dimer formation and interaction with BV/ODV-C42 and FP25. The charged domain was also required for BV/ODV-C42 interaction.

  13. Alternative splicing of a cryptic exon embedded in intron 6 of SMN1 and SMN2

    PubMed Central

    Yoshimoto, Satomi; Harahap, Nur Imma Fatimah; Hamamura, Yuko; Ar Rochmah, Mawaddah; Shima, Ai; Morisada, Naoya; Shinohara, Masakazu; Saito, Toshio; Saito, Kayoko; Lai, Poh San; Matsuo, Masafumi; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Nishio, Hisahide

    2016-01-01

    Both survival of motor neuron (SMN) genes are associated with spinal muscular atrophy; mutations in SMN1 cause the disease, and SMN2 modulates its severity. It is established that different alternative splicing of exon 7 occurs for SMN1 and SMN2, and a cryptic exon was recently found in intron 6 of both genes. Here, we characterize this cryptic exon and clarify its alternative splicing pattern in control and spinal muscular atrophy cells. PMID:27917293

  14. 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.

  15. Lex-SVM: exploring the potential of exon expression profiling for disease classification.

    PubMed

    Yuan, Xiongying; Zhao, Yi; Liu, Changning; Bu, Dongbo

    2011-04-01

    Exon expression profiling technologies, including exon arrays and RNA-Seq, measure the abundance of every exon in a gene. Compared with gene expression profiling technologies like 3' array, exon expression profiling technologies could detect alterations in both transcription and alternative splicing, therefore they are expected to be more sensitive in diagnosis. However, exon expression profiling also brings higher dimension, more redundancy, and significant correlation among features. Ignoring the correlation structure among exons of a gene, a popular classification method like L1-SVM selects exons individually from each gene and thus is vulnerable to noise. To overcome this limitation, we present in this paper a new variant of SVM named Lex-SVM to incorporate correlation structure among exons and known splicing patterns to promote classification performance. Specifically, we construct a new norm, ex-norm, including our prior knowledge on exon correlation structure to regularize the coefficients of a linear SVM. Lex-SVM can be solved efficiently using standard linear programming techniques. The advantage of Lex-SVM is that it can select features group-wisely, force features in a subgroup to take equal weihts and exclude the features that contradict the majority in the subgroup. Experimental results suggest that on exon expression profile, Lex-SVM is more accurate than existing methods. Lex-SVM also generates a more compact model and selects genes more consistently in cross-validation. Unlike L1-SVM selecting only one exon in a gene, Lex-SVM assigns equal weights to as many exons in a gene as possible, lending itself easier for further interpretation.

  16. Negative regulatory elements upstream of a novel exon of the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene.

    PubMed Central

    Bessis, A; Savatier, N; Devillers-Thiéry, A; Bejanin, S; Changeux, J P

    1993-01-01

    The expression of the nicotinic acetylcholine receptor alpha 2 subunit gene is highly restricted to the Spiriform lateralis nucleus of the Chick diencephalon. As a first step toward understanding the molecular mechanism underlying this regulation, we have investigated the structural and regulatory properties of the 5' sequence of this gene. A strategy based on the ligation of an oligonucleotide to the first strand of the cDNA (SLIC) followed by PCR amplification was used. A new exon was found approximately 3kb upstream from the first coding exon, and multiple transcription start sites of the gene were mapped. Analysis of the flanking region shows many consensus sequences for the binding of nuclear proteins, suggesting that the 1 kb flanking region contains at least a portion of the promoter of the gene. We have analysed the negative regulatory elements present within this region and found that a silencer region located between nucleotide -144 and +76 is active in fibroblasts as well as in neurons. This silencer is composed of six tandem repeat Oct-like motifs (CCCCATGCAAT), but does not bind any member of the Oct family. Moreover these motifs were found to act as a silencer only when they were tandemly repeated. When two, four or five motifs were deleted, the silencer activity of the motifs unexpectedly became an enhancer activity in all cells we have tested. Images PMID:8502560

  17. Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site.

    PubMed

    Otsuka, Hiroki; Sasai, Hideo; Nakama, Mina; Aoyama, Yuka; Abdelkreem, Elsayed; Ohnishi, Hidenori; Konstantopoulou, Vassiliki; Sass, Jörn Oliver; Fukao, Toshiyuki

    2016-11-01

    Beta-ketothiolase deficiency, also known as mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, is an autosomal recessive disease caused by mutations in the acetyl‑CoA acetyltransferase 1 (ACAT1) gene. A German T2‑deficient patient that developed a severe ketoacidotic episode at the age of 11 months, was revealed to be a compound heterozygote of a previously reported null mutation, c.472A>G (p.N158D) and a novel mutation, c.949G>A (p.D317N), in ACAT1. The c.949G>A mutation was suspected to cause aberrant splicing as it is located within an exonic splicing enhancer sequence (c. 947CTGACGC) that is a potential binding site for serine/arginine‑rich splicing factor 1. A mutation in this sequence, c.951C>T, results in exon 10 skipping. A minigene construct was synthesized that included exon 9‑truncated intron 9‑exon 10‑truncated intron 10‑exon 11, and the splicing of this minigene revealed that the c.949G>A mutant construct caused exon 10 skipping in a proportion of the transcripts. Furthermore, additional substitution of G for C at the first nucleotide of exon 10 (c.941G>C) abolished the effect of the c.949G>A mutation. Transient expression analysis of the c.949G>A mutant cDNA revealed no residual T2 activity in the mutated D317N enzyme. Therefore, c.949G>A (D317N) is a pathogenic missense mutation, and diminishes the effect of an exonic splicing enhancer and causes exon 10 skipping. The present study demonstrates that a missense mutation, or even a synonymous substitution, may disrupt enzyme function by interference with splicing.

  18. 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.

  19. Exon deletion patterns of the dystrophin gene in 82 Vietnamese Duchenne/Becker muscular dystrophy patients.

    PubMed

    Tran, Van Khanh; Ta, Van Thanh; Vu, Dung Chi; Nguyen, Suong Thi-Bang; Do, Hai Ngoc; Ta, Minh Hieu; Tran, Thinh Huy; Matsuo, Masafumi

    2013-12-01

    Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited muscle diseases caused by mutations in the dystrophin gene. The reading frame rule explains the genotype-phenotype relationship in DMD/BMD. In Vietnam, extensive mutation analysis has never been conducted in DMD/BMD. Here, 152 Vietnamese muscular dystrophy patients were examined for dystrophin exon deletion by amplifying 19 deletion-prone exons and deletion ends were confirmed by dystrophin cDNA analysis if necessary. The result was that 82 (54%) patients were found to have exon deletions, thus confirming exact deletion ends. A further result was that 37 patterns of deletion were classified. Deletions of exons 45-50 and 49-52 were the most common patterns identified, numbering six cases each (7.3%). The reading frame rule explained the genotype-phenotype relationship, but not five (6.1%) DMD cases. Each of five patients had deletions of exons 11-27 in common. The applicability of the therapy producing semifunctional in frame mRNA in DMD by inducing skipping of a single exon was examined. Induction of exon 51 skipping was ranked at top priority, since 16 (27%) patients were predicted to have semifunctional mRNA skipping. Exons 45 and 53 were the next ranked, with 12 (20%) and 11 (18%) patients, respectively. The largest deletion database of the dystrophin gene, established in Vietnamese DMD/BMD patients, disclosed a strong indication for exon-skipping therapy.

  20. Exon dosage analysis of parkin gene in Chinese sporadic Parkinson's disease.

    PubMed

    Guo, Ji-Feng; Dong, Xiao-Li; Xu, Qian; Li, Nan; Yan, Xin-Xiang; Xia, Kun; Tang, Bei-Sha

    2015-09-14

    Parkin gene mutations are by far the most common mutations in both familial Parkinson's disease (PD) and sporadic PD. Approximately, 50% of parkin mutations is exon dosage mutations (i.e., deletions and duplications of entire exons). Here, we first established a MLPA assay for quick detection of parkin exon rearrangements. Then, we studied parkin exon dosage mutations in 755 Chinese sporadic PDdisease patients using the established MLPA assay. We found that there were 25 (3.3%) patients with exon dosage alterations including deletions and duplications, 20 (11.4%) patients with exon rearrangements in 178 early-onset patients, and 5 (0.86%) patients with exon rearrangement mutations in 579 later-onset patients. The percentage of individuals with parkin dosage mutations is more than 33% when the age at onset is less than 30 years old, but less than 7% when the age at onset is more than 30. In these mutations, deletion is the main mutational style, especially in exon 2-5. Our results indicated that exon dosage mutations in parkin gene might be the main cause for sporadic PD, especially in EOP.

  1. An annotation infrastructure for the analysis and interpretation of Affymetrix exon array data.

    PubMed

    Okoniewski, Michał J; Yates, Tim; Dibben, Siân; Miller, Crispin J

    2007-01-01

    Affymetrix exon arrays contain probesets intended to target every known and predicted exon in the entire genome, posing significant challenges for high-throughput genome-wide data analysis. X:MAP http://xmap.picr.man.ac.uk, an annotation database, and exonmap http://www.bioconductor.org/packages/2.0/bioc/html/exonmap.html, a BioConductor/R package, are designed to support fine-grained analysis of exon array data. The system supports the application of standard statistical techniques, prior to the use of genome scale annotation to provide gene-, transcript- and exon-level summaries and visualization tools.

  2. An annotation infrastructure for the analysis and interpretation of Affymetrix exon array data

    PubMed Central

    Okoniewski, Michał J; Yates, Tim; Dibben, Siân; Miller, Crispin J

    2007-01-01

    Affymetrix exon arrays contain probesets intended to target every known and predicted exon in the entire genome, posing significant challenges for high-throughput genome-wide data analysis. X:MAP , an annotation database, and exonmap , a BioConductor/R package, are designed to support fine-grained analysis of exon array data. The system supports the application of standard statistical techniques, prior to the use of genome scale annotation to provide gene-, transcript- and exon-level summaries and visualization tools. PMID:17498294

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

    PubMed Central

    Sharma, Virag; Elghafari, Anas; Hiller, Michael

    2016-01-01

    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

  4. Exonic Re-Sequencing of the Chromosome 2q24.3 Parkinson’s Disease Locus

    PubMed Central

    Labbé, Catherine; Ogaki, Kotaro; Lorenzo-Betancor, Oswaldo; Carrasquillo, Minerva M.; Heckman, Michael G.; McCarthy, Allan; Soto-Ortolaza, Alexandra I.; Walton, Ronald L.; Lynch, Timothy; Siuda, Joanna; Opala, Grzegorz; Krygowska-Wajs, Anna; Barcikowska, Maria; Czyzewski, Krzysztof; Dickson, Dennis W.; Uitti, Ryan J.; Wszolek, Zbigniew K.; Ross, Owen A.

    2015-01-01

    Genome-wide association studies (GWAS) in Parkinson’s disease (PD) have identified over 20 genomic regions associated with disease risk. Many of these loci include several candidate genes making it difficult to pinpoint the causal gene. The locus on chromosome 2q24.3 encompasses three genes: B3GALT1, STK39, and CERS6. In order to identify if the causal variants are simple missense changes, we sequenced all 31 exons of these three genes in 187 patients with PD. We identified 13 exonic variants including four non-synonymous and three insertion/deletion variants (indels). These non-synonymous variants and rs2102808, the GWAS tag SNP, were genotyped in three independent series consisting of a total of 1976 patients and 1596 controls. Our results show that the seven identified 2q24.3 coding variants are not independently responsible for the GWAS association signal at the locus; however, there is a haplotype, which contains both rs2102808 and a STK39 exon 1 6bp indel variant, that is significantly associated with PD risk (Odds Ratio [OR] = 1.35, 95% CI: 1.11–1.64, P = 0.003). This haplotype is more associated than each of the two variants independently (OR = 1.23, P = 0.005 and 1.10, P = 0.10, respectively). Our findings suggest that the risk variant is likely located in a non-coding region. Additional sequencing of the locus including promoter and regulatory regions will be needed to pinpoint the association at this locus that leads to an increased risk to PD. PMID:26090850

  5. Exon Shuffling and Origin of Scorpion Venom Biodiversity

    PubMed Central

    Wang, Xueli; Gao, Bin; Zhu, Shunyi

    2016-01-01

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

  6. Exon Microarray Analysis of Human Dorsolateral Prefrontal Cortex in Alcoholism

    PubMed Central

    Manzardo, Ann M.; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G.

    2014-01-01

    Background Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Methods Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC, Brodmann area 9) of 7 adult Alcoholic (6 males, 1 female, mean age 48 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST Array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using qRT-PCR, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Results Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN) and signaling (e.g., RASGRP, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease, and development including cellular assembly and organization impacting on psychological disorders. Conclusions Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation and signaling that targets white matter of the brain. PMID:24890784

  7. 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

  8. 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.

  9. 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).

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

    PubMed Central

    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.

    2014-01-01

    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. PMID:24858907

  11. 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

  12. Unusual intron in the second exon of a Type III polyketide synthase gene of Alpinia calcarata Rosc.

    PubMed Central

    2010-01-01

    Plant phenolic compounds form a valuable resource of secondary metabolites having a broad spectrum of biological activities. Type III polyketide synthases play a key role in the formation of basic structural skeleton of the phenolic compounds. As a group of medicinal plants, PKSs with novel features are expected in the genome of Zingiberaceae. The genomic exploration of PKS in Alpinia calcarata conducted in this study identified the presence of an unusual intron at the region forming the second exon of typical PKSs, forming a gateway information of distribution of novel PKSs in Zingiberaceae. PMID:21637618

  13. 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

  14. 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.

  15. CDKN2A exon-wise deletion status and novel somatic mutations in Indian glioma patients.

    PubMed

    Sibin, M K; Bhat, Dhananjaya I; Lavanya, Ch; Manoj, M Jeru; Aakershita, S; Chetan, G K

    2014-02-01

    Over the years, deletions of CDKN2A (p16) tumor suppressor gene has been studied using FISH and multiplex PCR, with major focus on exon 2 in various cancers, and the frequency of mutation is found to be varied in different studies. In this study, we analyzed the deletion status of all three exons of p16 and frequency of exon 2 somatic point mutations in glioma from the Indian population and its clinical implications. Multiplex PCR was carried out in order to check deletion of all 3 exons in 50 glioma samples. Nonconventional PCR-SSCP analysis and sequencing was done to identify mutations in 48 cases. Deletion of at least one of the three exons of p16 INK4A was observed in ten cases (20 %). The frequencies of exon-wise deletions were 10 % for exon 1, 4 % for exon 2, and 8 % for exon 3. Two out of 48 samples were positive for mutations in p16 exon 2. One sample had a transition of G to C on position 147 with a codon change TGG to TGC which does not contribute to the protein structure. Another sample had a transversion of A to G on the position 154 with a codon change ATG to GTG with change in amino acid methionine to valine in 52nd position. Deletion pattern was found to be varied in three exons. Frequency of p16 gene mutation was less in the Indian population (4.2 %), and this mutation does not contribute to any remarkable change in protein structure.

  16. 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

  17. 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.

  18. 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.

  19. Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout

    PubMed Central

    Kapahnke, Marcel; Banning, Antje; Tikkanen, Ritva

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated sequence 9 (CRISPR/Cas9) system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level. PMID:27983621

  20. Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene.

    PubMed

    Aznarez, Isabel; Chan, Elayne M; Zielenski, Julian; Blencowe, Benjamin J; Tsui, Lap-Chee

    2003-08-15

    Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2alpha and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.

  1. Regulation of Dscam exon 17 alternative splicing by steric hindrance in combination with RNA secondary structures.

    PubMed

    Yue, Yuan; Li, Guoli; Yang, Yun; Zhang, Wenjing; Pan, Huawei; Chen, Ran; Shi, Feng; Jin, Yongfeng

    2013-12-01

    The gene Down syndrome cell adhesion molecule (Dscam) potentially encodes 38 016 distinct isoforms in Drosophila melanogaster via mutually exclusive splicing. Here we reveal a combinatorial mechanism of regulation of Dscam exon 17 mutually exclusive splicing through steric hindrance in combination with RNA secondary structure. This mutually exclusive behavior is enforced by steric hindrance, due to the close proximity of the exon 17.2 branch point to exon 17.1 in Diptera, and the interval size constraint in non-Dipteran species. Moreover, intron-exon RNA structures are evolutionarily conserved in 36 non-Drosophila species of six distantly related orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Phthiraptera), which regulates the selection of exon 17 variants via masking the splice site. By contrast, a previously uncharacterized RNA structure specifically activated exon 17.1 by bringing splice sites closer together in Drosophila, while the other moderately suppressed exon 17.1 selection by hindering the accessibility of polypyrimidine sequences. Taken together, these data suggest a phylogeny of increased complexity in regulating alternative splicing of Dscam exon 17 spanning more than 300 million years of insect evolution. These results also provide models of the regulation of alternative splicing through steric hindrance in combination with dynamic structural codes.

  2. SR proteins regulate V6 exon splicing of CD44 pre-mRNA.

    PubMed

    Loh, Tiing Jen; Moon, Heegyum; Jang, Ha Na; Liu, Yongchao; Choi, Namjeong; Shen, Shengfu; Williams, Darren Reece; Jung, Da-Woon; Zheng, Xuexiu; Shen, Haihong

    2016-11-01

    CD44 pre-mRNA includes 20 exons, of which exons 1-5 (C1-C5) and exons 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V6 splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V6 minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V6 splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V6 splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V6 specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V6-10 and V6,7-10 isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V6 splicing. [BMB Reports 2016; 49(11): 612-616].

  3. SR proteins regulate V6 exon splicing of CD44 pre-mRNA

    PubMed Central

    Loh, Tiing Jen; Moon, Heegyum; Jang, Ha Na; Liu, Yongchao; Choi, Namjeong; Shen, Shengfu; Williams, Darren Reece; Jung, Da-Woon; Zheng, Xuexiu; Shen, Haihong

    2016-01-01

    CD44 pre-mRNA includes 20 exons, of which exons 1–5 (C1–C5) and exons 16–20 (C6–C10) are constant exons, whereas exons 6–15 (V1–V10) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V6 splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V6 minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V6 splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V6 splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V6 specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V6–10 and V6,8–10 isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V6 splicing. PMID:27530682

  4. 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.

  5. Exon-level expression analyses identify MYCN and NTRK1 as major determinants of alternative exon usage and robustly predict primary neuroblastoma outcome

    PubMed Central

    Schramm, A; Schowe, B; Fielitz, K; Heilmann, M; Martin, M; Marschall, T; Köster, J; Vandesompele, J; Vermeulen, J; de Preter, K; Koster, J; Versteeg, R; Noguera, R; Speleman, F; Rahmann, S; Eggert, A; Morik, K; Schulte, J H

    2012-01-01

    Background: Using mRNA expression-derived signatures as predictors of individual patient outcome has been a goal ever since the introduction of microarrays. Here, we addressed whether analyses of tumour mRNA at the exon level can improve on the predictive power and classification accuracy of gene-based expression profiles using neuroblastoma as a model. Methods: In a patient cohort comprising 113 primary neuroblastoma specimens expression profiling using exon-level analyses was performed to define predictive signatures using various machine-learning techniques. Alternative transcript use was calculated from relative exon expression. Validation of alternative transcripts was achieved using qPCR- and cell-based approaches. Results: Both predictors derived from the gene or the exon levels resulted in prediction accuracies >80% for both event-free and overall survival and proved as independent prognostic markers in multivariate analyses. Alternative transcript use was most prominently linked to the amplification status of the MYCN oncogene, expression of the TrkA/NTRK1 neurotrophin receptor and survival. Conclusion: As exon level-based prediction yields comparable, but not significantly better, prediction accuracy than gene expression-based predictors, gene-based assays seem to be sufficiently precise for predicting outcome of neuroblastoma patients. However, exon-level analyses provide added knowledge by identifying alternative transcript use, which should deepen the understanding of neuroblastoma biology. PMID:23047593

  6. Alternative splicing and evolution: diversification, exon definition and function.

    PubMed

    Keren, Hadas; Lev-Maor, Galit; Ast, Gil

    2010-05-01

    Over the past decade, it has been shown that alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, particularly in mammals. Splicing can be found in species from bacteria to humans, but its prevalence and characteristics vary considerably. Evolutionary studies are helping to address questions that are fundamental to understanding this important process: how and when did AS evolve? Which AS events are functional? What are the evolutionary forces that shaped, and continue to shape, AS? And what determines whether an exon is spliced in a constitutive or alternative manner? In this Review, we summarize the current knowledge of AS and evolution and provide insights into some of these unresolved questions.

  7. Modeling the evolution dynamics of exon-intron structure with a general random fragmentation process

    PubMed Central

    2013-01-01

    Background Most eukaryotic genes are interrupted by spliceosomal introns. The evolution of exon-intron structure remains mysterious despite rapid advance in genome sequencing technique. In this work, a novel approach is taken based on the assumptions that the evolution of exon-intron structure is a stochastic process, and that the characteristics of this process can be understood by examining its historical outcome, the present-day size distribution of internal translated exons (exon). Through the combination of simulation and modeling the size distribution of exons in different species, we propose a general random fragmentation process (GRFP) to characterize the evolution dynamics of exon-intron structure. This model accurately predicts the probability that an exon will be split by a new intron and the distribution of novel insertions along the length of the exon. Results As the first observation from this model, we show that the chance for an exon to obtain an intron is proportional to its size to the 3rd power. We also show that such size dependence is nearly constant across gene, with the exception of the exons adjacent to the 5′ UTR. As the second conclusion from the model, we show that intron insertion loci follow a normal distribution with a mean of 0.5 (center of the exon) and a standard deviation of 0.11. Finally, we show that intron insertions within a gene are independent of each other for vertebrates, but are more negatively correlated for non-vertebrate. We use simulation to demonstrate that the negative correlation might result from significant intron loss during evolution, which could be explained by selection against multi-intron genes in these organisms. Conclusions The GRFP model suggests that intron gain is dynamic with a higher chance for longer exons; introns are inserted into exons randomly with the highest probability at the center of the exon. GRFP estimates that there are 78 introns in every 10 kb coding sequences for vertebrate genomes

  8. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD.

    PubMed

    Ling, Jonathan P; Pletnikova, Olga; Troncoso, Juan C; Wong, Philip C

    2015-08-07

    Cytoplasmic aggregation of TDP-43, accompanied by its nuclear clearance, is a key common pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, a limited understanding of this RNA-binding protein (RBP) impedes the clarification of pathogenic mechanisms underlying TDP-43 proteinopathy. In contrast to RBPs that regulate splicing of conserved exons, we found that TDP-43 repressed the splicing of nonconserved cryptic exons, maintaining intron integrity. When TDP-43 was depleted from mouse embryonic stem cells, these cryptic exons were spliced into messenger RNAs, often disrupting their translation and promoting nonsense-mediated decay. Moreover, enforced repression of cryptic exons prevented cell death in TDP-43-deficient cells. Furthermore, repression of cryptic exons was impaired in ALS-FTD cases, suggesting that this splicing defect could potentially underlie TDP-43 proteinopathy.

  9. 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.

  10. Identification of NURR1 (Exon 4) and FOXA1 (Exon 3) Haplotypes Associated with mRNA Expression Levels in Peripheral Blood Lymphocytes of Parkinson's Patients in Small Indian Population

    PubMed Central

    Tippabathani, Jayakrishna; Radhakrishnan, Vaishnavie; Banik, Somashree; Kapoor, Sonia

    2017-01-01

    Here, we study the expression of NURR1 and FOXA1 mRNA in peripheral blood lymphocytes and its haplotypes in coding region in a small Chennai population of India. Thirty cases of Parkinson's patients (PD) with anti-PD medications (20 males aged 65.85 ± 1.19 and 10 females aged 65.7 ± 1.202) and 30 age matched healthy people (20 males aged 68.45 ± 1.282 and 10 females aged 65.8 ± 1.133) were included. The expression of NURR1 and FOXA1 in PBL was detected by Q-PCR and haplotypes were identified by PCR-SSCP. In the 30 PD cases examined, NURR1 and FOXA1 expression was significantly reduced in both male and female PD patients. However, NURR1 (57.631% reduced in males; 28.93% in females) and FOXA1 (64.42% in males; 55.76% in females) mRNA expression did differ greatly between male and female PD patients. Polymorphisms were identified at exon 4 of the NURR1 and at exon 3 of the FOXA1, respectively, in both male and female patients. A near significant difference in SSCP patterns between genders of control and PD population was analyzed suggesting that further investigations of more patients, more molecular markers, and coding regions should be performed. Such studies could potentially reveal peripheral molecular marker of early PD and different significance to the respective genders. PMID:28255498

  11. Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization

    PubMed Central

    Eissa, N. Tony; Yuan, Jean W.; Haggerty, Cynthia M.; Choo, Esther K.; Palmer, Celeste D.; Moss, Joel

    1998-01-01

    The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242–335 of the oxygenase domain. In this study, iNOS8−9− and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8−9− in 293 cell line resulted in generation of iNOS8−9− mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8−9− did not form dimers. Similar to iNOSFL, iNOS8−9− exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two “control” iNOS deletion mutants were synthesized, lacking either residues 45–52 of the oxygenase domain or residues 1131–1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis. PMID:9636200

  12. 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

  13. Identification of beta1C-2, a novel variant of the integrin beta1 subunit generated by utilization of an alternative splice acceptor site in exon C.

    PubMed Central

    Svineng, G; Fässler, R; Johansson, S

    1998-01-01

    A new splice variant of the human integrin subunit beta1 has been identified and designated beta1C-2. It differs from the previously reported beta1C (in this report designated beta1C-1) by 18 nucleotides, and is generated by splicing from exon 6 to an alternative splice acceptor site within exon C, causing an in-frame deletion of six amino acids of the cytoplasmic region of beta1C-1. The beta1C-2 mRNA is present in several human cell lines and tissues at low levels, similarly to beta1C-1. In peripheral T-lymphocytes, beta1C-2 is the selectively expressed isoform. Neither beta1C-1 nor beta1C-2 mRNA could be detected in mouse tissues, and Southern hybridization of a mouse genomic beta1 clone with a human exon-C-specific probe failed to identify a corresponding mouse exon. The antisense orientation of exon C is highly homologous to an Alu element. Since Alu elements are restricted to primates, the beta1C-1 and beta1C-2 variants of the integrin subunit beta1 are specific for these species. The protein coded for by the beta1C-2 cDNA can be expressed and localized to the surface of beta1 deficient mouse cells. However, while stable transformed clones expressing high levels of the beta1A were commonly found, the beta1C-1 and beta1C-2 expressing clones expressed barely detectable amounts of the beta1 protein. Hence, high levels of beta1C-2 may be incompatible with cell proliferation, as previously suggested for beta1C-1. PMID:9494094

  14. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

    PubMed Central

    Balestra, Dario; Scalet, Daniela; Pagani, Franco; Rogalska, Malgorzata Ewa; Mari, Rosella; Bernardi, Francesco; Pinotti, Mirko

    2016-01-01

    In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients. PMID:27701399

  15. THE CRYSTAL STRUCTURE OF 2,7-DIACETOXYTRANS-15,16-DIMETHYL-15,16-DIHYDROPYRENE,

    DTIC Science & Technology

    AROMATIC COMPOUNDS, CRYSTAL STRUCTURE ), (*POLYCYCLIC COMPOUNDS, CRYSTAL STRUCTURE ), (* CRYSTAL STRUCTURE , POLYCYCLIC COMPOUNDS), ESTERS, MOLECULAR STRUCTURE, CHEMICAL BONDS, X RAY DIFFRACTION, SCINTILLATION COUNTERS, CANADA

  16. Alternative splicing of U2AF1 reveals a shared repression mechanism for duplicated exons

    PubMed Central

    Kralovicova, Jana; Vorechovsky, Igor

    2017-01-01

    The auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF) facilitates branch point (BP) recognition and formation of lariat introns. The gene for the 35-kD subunit of U2AF gives rise to two protein isoforms (termed U2AF35a and U2AF35b) that are encoded by alternatively spliced exons 3 and Ab, respectively. The splicing recognition sequences of exon 3 are less favorable than exon Ab, yet U2AF35a expression is higher than U2AF35b across tissues. We show that U2AF35b repression is facilitated by weak, closely spaced BPs next to a long polypyrimidine tract of exon Ab. Each BP lacked canonical uridines at position -2 relative to the BP adenines, with efficient U2 base-pairing interactions predicted only for shifted registers reminiscent of programmed ribosomal frameshifting. The BP cluster was compensated by interactions involving unpaired cytosines in an upstream, EvoFold-predicted stem loop (termed ESL) that binds FUBP1/2. Exon Ab inclusion correlated with predicted free energies of mutant ESLs, suggesting that the ESL operates as a conserved rheostat between long inverted repeats upstream of each exon. The isoform-specific U2AF35 expression was U2AF65-dependent, required interactions between the U2AF-homology motif (UHM) and the α6 helix of U2AF35, and was fine-tuned by exon Ab/3 variants. Finally, we identify tandem homologous exons regulated by U2AF and show that their preferential responses to U2AF65-related proteins and SRSF3 are associated with unpaired pre-mRNA segments upstream of U2AF-repressed 3′ss. These results provide new insights into tissue-specific subfunctionalization of duplicated exons in vertebrate evolution and expand the repertoire of exon repression mechanisms that control alternative splicing. PMID:27566151

  17. 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.

  18. BEAT: Bioinformatics Exon Array Tool to store, analyze and visualize Affymetrix GeneChip Human Exon Array data from disease experiments

    PubMed Central

    2012-01-01

    Background It is known from recent studies that more than 90% of human multi-exon genes are subject to Alternative Splicing (AS), a key molecular mechanism in which multiple transcripts may be generated from a single gene. It is widely recognized that a breakdown in AS mechanisms plays an important role in cellular differentiation and pathologies. Polymerase Chain Reactions, microarrays and sequencing technologies have been applied to the study of transcript diversity arising from alternative expression. Last generation Affymetrix GeneChip Human Exon 1.0 ST Arrays offer a more detailed view of the gene expression profile providing information on the AS patterns. The exon array technology, with more than five million data points, can detect approximately one million exons, and it allows performing analyses at both gene and exon level. In this paper we describe BEAT, an integrated user-friendly bioinformatics framework to store, analyze and visualize exon arrays datasets. It combines a data warehouse approach with some rigorous statistical methods for assessing the AS of genes involved in diseases. Meta statistics are proposed as a novel approach to explore the analysis results. BEAT is available at http://beat.ba.itb.cnr.it. Results BEAT is a web tool which allows uploading and analyzing exon array datasets using standard statistical methods and an easy-to-use graphical web front-end. BEAT has been tested on a dataset with 173 samples and tuned using new datasets of exon array experiments from 28 colorectal cancer and 26 renal cell cancer samples produced at the Medical Genetics Unit of IRCCS Casa Sollievo della Sofferenza. To highlight all possible AS events, alternative names, accession Ids, Gene Ontology terms and biochemical pathways annotations are integrated with exon and gene level expression plots. The user can customize the results choosing custom thresholds for the statistical parameters and exploiting the available clinical data of the samples for a

  19. 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.

  20. 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.

  1. EGFR exon mutation distribution and outcome in non-small-cell lung cancer: a Portuguese retrospective study.

    PubMed

    de Mello, Ramon Andrade; Pires, Filipa Soares; Marques, Dânia Sofia; Oliveira, Júlio; Rodrigues, Ana; Soares, Marta; Azevedo, Isabel; Peixoto, Ana; Santos, Catarina; Pinto, Carla; Hespanhol, Venceslau; Teixeira, Manuel R; Amaro, Teresina; Queiroga, Henrique; Araújo, António

    2012-12-01

    Epidermal growth factor receptor (EGFR) mutations play a predictive role in advanced stages of non-small-cell lung cancer (NSCLC) patients. We conducted this study in order to assess EGFR status in a Portuguese population and its role in NSCLC patients' outcomes. Patients were submitted to EGFR assessment by high-resolution melting and/or direct sequencing. Kaplan-Meier curve was used to assess overall survival and progression-free survival (PFS). Two hundred forty eight out of 322 participants were assessed for EGFR status. Forty-two patients (16.9 %) presented EGFR-mutated status: one patient (2.4 %) presented exon 18; 21 patients (50 %), exon 19; one patient (2.4 %), exon 20; and 18 patients (45.2 %), exon 21 mutations, p < 0.001. PFS was not assessed (n.a.) for patient with exon 18 mutation, and for the other patients with mutations, it was 7 months (3.96-10.03) (exon 19), <1 month (exon 20), and 7 months (0-14.2) (exon 21) (p = 0.027). Overall survival (OS) was 11 months (exon 18), 11 months (1-18) (exon 19), 1 month (exon 20), and 7.5 months (2-70) (exon 21) (p = n.a). This study suggests that the EGFR mutation is herein observed in a higher proportion than expected for a Caucasian population, and OS is a little less than that published in the literature.

  2. Pattern recognition in DNA sequences: The intron-exon junction problem

    SciTech Connect

    Mural, R.J.; Uberbacher, E.C. Tennessee Univ., Oak Ridge, TN . Graduate School of Biomedical Sciences); Mann, R.C. )

    1990-01-01

    One of the fundamental problems facing the field of genomic sequence analysis is the difficulty in locating relatively small coding regions of DNA within the much larger non-coding regions. Neural networks, linguistic analysis and various types of expert systems have been used with various degrees of success to address this problem. We have developed several methods for recognizing the presence of splice junctions and coding DNA which are based on artificial intelligence, linguistic and statistical approaches. The triplet vocabulary in and around splice junctions has been analyzed for primates, and the occurrences of preferred triplets in potential junctions seems to be a very selective method for distinguishing true junctions from otherwise similar sequences. given a 50% mix of true and false junctions, this method scores 93%--95% correct. Several approaches have been used to identify exons. These include a frame bias matrix algorithm and an algorithm which estimates the fractal dimension of dinucleotide usage. Attempts are underway to combine the outputs of the various methods using a rule-based approach to improve the overall performance of these predictors. 13 refs., 4 figs.

  3. Characterization of 5' promoter and exon 1-3 polymorphism of the RAET1E gene.

    PubMed

    Cox, Steven T; Pearson, Hayley; Laza-Briviesca, Raquel; Pesoa, Susanna; Vullo, Carlos; Madrigal, J Alejandro; Saudemont, Aurore

    2016-01-01

    NKG2D is an activating receptor utilized by natural killer (NK) cells that recognizes upregulated ligands on infected, tumorigenic and damaged cells, leading to their cytolysis. However, the NKG2D ligand (NKG2DL) system is very complex with eight known gene loci encoding slightly different molecules. Furthermore, most NKG2DL gene loci such as MICA and MICB are highly polymorphic with potential for functional differences. NKG2DL expression on tumors varies depending on the malignancy and tumors can also release soluble NKG2DL that exert anergic effects on NK cells when engagement with NKG2D occurs, allowing escape from NK cell immunosurveillance. We carried out RAET1E typing of IHW cell line DNA, including a 580 bp proximal promoter fragment and exons 1-3 identifying 13 of 15 known RAET1E alleles. We determined 7 polymorphisms within the promoter region, including 2 already known that contributed to 9 promoter types. RAET1E alleles with variability in the extracellular region also differed with respect to promoter type and one allele, RAET1E(∗)003, associated with 5 promoter types. We then identified putative transcription factor binding sites for RAET1E, and found 5 of the 7 promoter polymorphisms may disrupt these sites, abrogating binding of transcription factors and varying the potential level of expression.

  4. Does the mutant CAG expansion in huntingtin mRNA interfere with exonucleolytic cleavage of its first exon?

    PubMed Central

    Liu, Wanzhao; Pfister, Edith L.; Kennington, Lori A.; Chase, Kathryn O.; Mueller, Christian; DiFiglia, Marian; Aronin, Neil

    2016-01-01

    Background Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington’s disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments. Objectives We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA. Methods Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection. Results Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites. Conclusions Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage. PMID:27003665

  5. Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein*

    PubMed Central

    Nucifora, Leslie G.; Burke, Kathleen A.; Feng, Xia; Arbez, Nicolas; Zhu, Shanshan; Miller, Jason; Yang, Guocheng; Ratovitski, Tamara; Delannoy, Michael; Muchowski, Paul J.; Finkbeiner, Steven; Legleiter, Justin; Ross, Christopher A.; Poirier, Michelle A.

    2012-01-01

    Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity. PMID:22433867

  6. 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.

  7. Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer.

    PubMed

    Yasuda, Hiroyuki; Park, Eunyoung; Yun, Cai-Hong; Sng, Natasha J; Lucena-Araujo, Antonio R; Yeo, Wee-Lee; Huberman, Mark S; Cohen, David W; Nakayama, Sohei; Ishioka, Kota; Yamaguchi, Norihiro; Hanna, Megan; Oxnard, Geoffrey R; Lathan, Christopher S; Moran, Teresa; Sequist, Lecia V; Chaft, Jamie E; Riely, Gregory J; Arcila, Maria E; Soo, Ross A; Meyerson, Matthew; Eck, Michael J; Kobayashi, Susumu S; Costa, Daniel B

    2013-12-18

    Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R, and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (~10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib, and afatinib). The basis of this primary resistance is poorly understood. We studied a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients, and found that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered adenosine triphosphate-binding pocket, and the inserted residues form a wedge at the end of the C helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro, and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology, and their response to EGFR TKIs.

  8. 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.

  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

    2015-11-02

    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.

  10. FUNCTIONAL UNDERSTANDING OF THE DIVERSE EXON-INTRON STRUCTURES OF HUMAN GPCR GENES

    PubMed Central

    HAMMOND, DOROTHY A.; OLMAN, VICTOR

    2014-01-01

    The GPCR genes have a variety of exon-intron structures even though their proteins are all structurally homologous. We have examined all human GPCR genes with at least two functional protein isoforms, totaling 199, aiming to gain an understanding of what may have contributed to the large diversity of the exon-intron structures of the GPCR genes. The 199 genes have a total of 808 known protein splicing isoforms with experimentally verified functions. Our analysis reveals that 1,301 (80.6%) adjacent exon-exon pairs out of the total of 1,613 in the 199 genes have either exactly one exon skipped or the intron in-between retained in at least one of the 808 protein splicing isoforms. This observation has a statistical significance p-value of 2.051762* e−09, assuming that the observed splicing isoforms are independent of the exon-intron structures. Our interpretation of this observation is that the exon boundaries of the GPCR genes are not randomly determined; instead they may be selected to facilitate specific alternative splicing for functional purposes. PMID:24467758

  11. 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

  12. A novel insertion mutation on exon 20 of epidermal growth factor receptor, conferring resistance to erlotinib.

    PubMed

    Khan, Nawazish A; Mirshahidi, Saied; Mirshahidi, Hamid R

    2014-05-01

    The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein tyrosine kinase receptor. The small-molecule tyrosine kinase receptor inhibitors (TKIs) are in clinical use to treat non-small cell lung cancer with EGFR mutations. Variable tumor responses to erlotinib and gefitinib have been observed. The response to these TKIs varies by the type of EGFR mutations found in the tumor. The deletion on exon 19 and the L858R substitution on exon 21 constitute the most frequent mutations and are known to show good response to TKIs. However, mutations on exon 20 are less common and seem to respond poorly to TKIs. In clinical settings, the reported response of exon 20 mutations to reversible TKIs (both gefitinib and erlotinib) remains inconstant. The type of coexisting mutation seems to affect the response of these insertions to TKIs. We herein present a case of disease progression despite the use of erlotinib in a female patient who had a novel insertion mutation on exon 20. Our patient was a never-smoker and was identified to have a Pro772_His773insGlnCysPro mutation on exon 20. She had previously been treated with cisplatin and gemcitabine and then with carboplatin and pemetrexed. She was treated with erlotinib upon intolerance to second-line chemotherapy and did not respond. Our patient had a novel insertion mutation on exon 20, which was found to be resistant to erlotinib.

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

    PubMed Central

    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.

    2015-01-01

    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

  14. 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.

  15. The evolutionary fate of alternatively spliced homologous exons after gene duplication.

    PubMed

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

    2015-04-29

    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.

  16. DNA aptamers against exon v10 of CD44 inhibit breast cancer cell migration.

    PubMed

    Iida, Joji; Clancy, Rebecca; Dorchak, Jesse; Somiari, Richard I; Somiari, Stella; Cutler, Mary Lou; Mural, Richard J; Shriver, Craig D

    2014-01-01

    CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10.

  17. Exon-skipped dystrophins for treatment of Duchenne muscular dystrophy: mass spectrometry mapping of most exons and cooperative domain designs based on single molecule mechanics.

    PubMed

    Krieger, Christine Carag; Bhasin, Nishant; Tewari, Manorama; Brown, Andre E X; Safer, Daniel; Sweeney, H Lee; Discher, Dennis E

    2010-12-01

    Force-bearing linkages between the cytoskeleton and extracellular matrix are clearly important to normal cell viability-as is evident in a disease such as Duchenne muscular dystrophy (DMD) which arises in the absence of the linkage protein dystrophin. Therapeutic approaches to DMD include antisense-mediated skipping of exons to delete nonsense mutations while maintaining reading frame, but the structure and stability of the resulting proteins are generally unclear. Here we use mass spectrometry to detect most dystrophin exons, and we express and physically characterize dystrophin "nano"-constructs based on multiexon deletions that might find use in a large percentage of DMD patients. The primary structure challenge is addressed first with liquid chromatography tandem mass spectrometry (LC-MS/MS) which can detect tryptic peptides from 53 of dystrophin's 79 exons; equivalent information from immunodetection would require 53 different high-specificity antibodies. Folding predictions for the nano-constructs reveal novel helical bundle domains that arise out of exon-deleted "linkers," while secondary structure studies confirm high helicity and also melting temperatures well above physiological. Extensional forces with an atomic force microscope nonetheless unfold the constructs, and the ensemble of unfolding trajectories reveal the number of folded domains, proving consistent with structure predictions. A mechanical cooperativity parameter for unfolding of tandem domains is also introduced as the best predictor of a multiexon deletion that is asymptomatic in humans. The results thereby provide insight and confidence in exon-skipped designs.

  18. Identification of a new complex rearrangement affecting exon 20 of BRCA1.

    PubMed

    Del Valle, Jesús; Campos, Olga; Velasco, Angela; Darder, Esther; Menéndez, Mireia; Feliubadaló, Lídia; Tornero, Eva; Blanco, Ignacio; Izquierdo, Angel; Brunet, Joan; Capellá, Gabriel; Lázaro, Conxi

    2011-11-01

    In this study, we present a novel complex rearrangement in the BRCA1 gene. The genomic rearrangement was identified using one of the two commercially available MLPA BRCA1 kits but was not confirmed with the other. In this report, we present the full characterization at the DNA and RNA levels of a new partial deletion of exon 20 of BRCA1. This is a complex deletion with four breakpoints which promotes aberrant splicing with partial deletion of exon 20 plus the insertion of a cryptic exon corresponding to a fragment of intron 20. The aberrant splicing generates an abnormal transcript with a frameshift that will result in a truncated BRCA1 protein.

  19. 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.

  20. p53 exon 7 mutations as a predictor of poor prognosis in patients with colorectal cancer.

    PubMed

    Iniesta, P; Vega, F J; Caldés, T; Massa, M; de Juan, C; Cerdán, F J; Sánchez, A; López, J A; Torres, A J; Balibrea, J L; Benito, M

    1998-08-14

    We have studied 61 resected colorectal adenocarcinomas in order to investigate p53 mutations as a prognostic factor for this pathology. Mutations in exons 5-9 of the p53 gene were analyzed by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique followed by sequencing. Our data indicate that p53 exon 7 mutations were prevalent in the latest stages of colorectal carcinogenesis and patients bearing this alteration had the worst prognosis. Therefore, according to our results, mutations affecting exon 7 of the p53 gene could be considered as a useful marker of biological aggressiveness for colorectal cancer.

  1. 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

  2. A novel intronic cis element, ISE/ISS-3, regulates rat fibroblast growth factor receptor 2 splicing through activation of an upstream exon and repression of a downstream exon containing a noncanonical branch point sequence.

    PubMed

    Hovhannisyan, Ruben H; Carstens, Russ P

    2005-01-01

    Mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc yields two receptor isoforms, FGFR2-IIIb and -IIIc, with distinctly different ligand binding properties. Several RNA cis elements in the intron (intron 8) separating these exons have been described that are required for splicing regulation. Using a heterologous splicing reporter, we have identified a new regulatory element in this intron that confers cell-type-specific inclusion of an unrelated exon that mirrors its ability to promote cell-type-specific inclusion of exon IIIb. This element promoted inclusion of exon IIIb while at the same time silencing exon IIIc inclusion in cells expressing FGFR2-IIIb; hence, we have termed this element ISE/ISS-3 (for "intronic splicing enhancer-intronic splicing silencer 3"). Silencing of exon IIIc splicing by ISE/ISS-3 was shown to require a branch point sequence (BPS) using G as the primary branch nucleotide. Replacing a consensus BPS with A as the primary branch nucleotide resulted in constitutive splicing of exon IIIc. Our results suggest that the branch point sequence constitutes an important component that can contribute to the efficiency of exon definition of alternatively spliced cassette exons. Noncanonical branch points may thus facilitate cell-type-specific silencing of regulated exons by flanking cis elements.

  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. 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.

  5. Hereditary sideroblastic anaemia due to a mutation in exon 10 of the erythroid 5-aminolaevulinate synthase gene.

    PubMed

    Edgar, A J; Wickramasinghe, S N

    1998-02-01

    DNA sequencing of the coding region of the erythroid 5-aminolaevulinate synthase (ALAS2) cDNA from a male with pyridoxine-responsive sideroblastic anaemia revealed a missense mutation C1622G and a closely linked polymorphism C1612A in exon 10 of the gene. Sequence analysis of the genomic DNA from other family members revealed that the proband's mother and daughter were heterozygous carriers of the mutation, consistent with the X-linked inheritance. The C1622G mutation results in a histidine to aspartic acid substitution at amino acid residue 524. The histidine residue is conserved in both the erythroid and housekeeping ALAS proteins in vertebrates, all other known ALAS proteins and other oxamine synthases that have pyridoxal 5'-phosphate as a co-factor. This histidine is located in a predicted loop, preceding a long alpha-helix region near the carboxy-terminus.

  6. De novo exonic mutation in MYH7 gene leading to exon skipping in a patient with early onset muscular weakness and fiber-type disproportion.

    PubMed

    Pajusalu, Sander; Talvik, Inga; Noormets, Klari; Talvik, Tiina; Põder, Haide; Joost, Kairit; Puusepp, Sanna; Piirsoo, Andres; Stenzel, Werner; Goebel, Hans H; Nikopensius, Tiit; Annilo, Tarmo; Nõukas, Margit; Metspalu, Andres; Õunap, Katrin; Reimand, Tiia

    2016-03-01

    Here we report on a case of MYH7-related myopathy in a boy with early onset of muscular weakness and delayed motor development in infancy. His most affected muscles were neck extensors showing a dropped head sign, proximal muscles of lower limbs with positive Gower's sign, and trunk muscles. Brain and spinal cord MRI scans, echocardiography, and laboratory analyses including creatine kinase and lactate did not reveal any abnormalities. Muscle histopathology showed fiber-type disproportion. Whole exome sequencing of the parents-offspring trio revealed a novel de novo c.5655G>A p.(Ala1885=) synonymous substitution of the last nucleotide in exon 38 of the MYH7 gene. Further RNA investigations proved the skipping of exon 38 (p.1854_1885del). This is a first report of an exon-skipping mutation in the MYH7 gene causing myopathy. This report broadens both the phenotypic and genotypic spectra of MYH7-related myopathies.

  7. WT1 exon deletion/insertion mutations in Wilms tumor patients associated with di- and trinucleotide repeats and deletion hotspot consensus sequences

    SciTech Connect

    Huff, V.; Saunders, G.F.; Strong, L.C.

    1994-09-01

    The genetic etiology of Wilms tumor (WT), a childhood kidney tumor, is known to be heterogeneous. One WT gene, WT1 located on chromosomal band 11p13, has been isolated, and mutations specific to the WT1 locus have been identified in some WT patients, demonstrating its importance in the etiology of at least some WT cases. Because of the patient populations selected for study and a focus on specific regions of the gene in identifying WT1 mutations, most mutations described to date are single base substitutions and have occurred in the 3{prime} portion of the gene in exons 7-10 which encode the zinc finger domains of the protein. DNA mutations at the WT1 locus have been reported to be infrequent in WT patients, implying that post-transcriptional modification of the WT1 gene product or alterations at other distinct loci perhaps more commonly play a role in tumorigenesis than do WT1 DNA mutations. We report here the occurrence in four WT patients of inactivating frameshift mutations, three somatic deletions and one germline insertion, in exon 1. This exon is GC-rich (>70%) and contains regions of both di- and trinucleotide direct repeats and ten copies of the sequence, CCTG (CAGG), which has been described as a deletion hotspot in mammalian DNA. The four WT1 exon 1 deletion/insertions range in size from four to 34 bases, are flanked by bi- and/or trinucleotide repeats, and occur in a general context of repetitive sequences. Furthermore, all breakpoints occur at or within five nucleotides of the tetranucleotide sequence, CCTG (CAGG). These data suggest that a mutational mechanism previously unrecognized in WT patients is operating in this repetitive portion of the WT1 gene. The frequency with which mutations of this type are observed in WT patients awaits the complete evaluation of a series of patients. Nevertheless these data indicate that WT1 mutations are more frequent in WT patients than previously thought.

  8. hnRNP L inhibits CD44 V10 exon splicing through interacting with its upstream intron.

    PubMed

    Loh, Tiing Jen; Cho, Sunghee; Moon, Heegyum; Jang, Ha Na; Williams, Darren Reece; Jung, Da-Woon; Kim, Il-Chul; Ghigna, Claudia; Biamonti, Giuseppe; Zheng, Xuexiu; Shen, Haihong

    2015-06-01

    CD44 is a complex cell adhesion molecule that mediates communication and adhesion between adjacent cells as well as between cells and the extracellular matrix. CD44 pre-mRNA produces various mRNA isoforms through alternative splicing of 20 exons, among which exons 1-5 (C1-C5) and 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. CD44 V10 exon has important roles in breast tumor progression and Hodgkin lymphoma. Here we show that increased expression of hnRNP L inhibits V10 exon splicing of CD44 pre-mRNA, whereas reduced expression of hnRNP L promotes V10 exon splicing. In addition, hnRNP L also promotes V10 splicing of endogenous CD44 pre-mRNA. Through mutation analysis, we demonstrate that the effects of hnRNP L on V10 splicing are abolished when the CA-rich sequence on the upstream intron of V10 exon is disrupted. However, hnRNP L effects are stronger if more CA-repeats are provided. Furthermore, we show that hnRNP L directly contacts the CA-rich sequence. Importantly, we provide evidences that hnRNP L inhibits U2AF65 binding on the upstream Py tract of V10 exon. Our results reveal that hnRNP L is a new regulator for CD44 V10 exon splicing.

  9. Origin and evolution of a new exon of 14-3-3ξ in bees and phylogenetic analysis.

    PubMed

    Zhan, Leilei; Jiang, Chao

    2013-04-01

    Mutually exclusive splicing, one type of alternative splicing, involves selection of alternatively spliced exons arranged in tandem and creates protein products with substitution of one segment of the amino acid sequence for another. Previous studies revealed that exon 5 of 14-3-3ξ from Apis mellifera (western honeybee) had three mutually exclusive exons, while orthologous exon of Nasonia vitripennis (parasitic wasp) had only two, suggesting that cases of exon gain or loss might have happened during the evolution of hymenopteran species. In the current study, we annotated and analyzed the 14-3-3ξ genes from 20 hymenopteran species successfully, and the results of phylogenetic analysis revealed the presence of a new mutually exclusive exon in corbiculate bees. In addition, we found that duplication via staggered homologous recombination was responsible for the origin of the new exon.

  10. Characterization of rat c-myc and adjacent regions.

    PubMed Central

    Hayashi, K; Makino, R; Kawamura, H; Arisawa, A; Yoneda, K

    1987-01-01

    Rat genomic regions covering c-myc were cloned from the DNA of both normal liver and two lines of Morris hepatomas, one of which had c-myc amplification. The three restriction maps showed perfect agreement within the overlapping regions. The 7 kb regions, which included the entire normal rat c-myc and the region 2.2 kb upstream, and one from the hepatomas, were sequenced and found to be identical. The coding regions of exons 2 and 3 were highly conserved between rat, mouse and man, but some differences in amino acids were noted. Exon 1 and the non-coding region of exon 3 showed limited homology between the three species. Rat exon 1 contained several nonsense codons in each frame and no ATG codon, indicating there to be no coding capacity in this exon. The 2.2 kb upstream regions and the introns compared showed unusual conservation between the rat and human genes. Some motifs, previously proposed as having a functional role in human c-myc, were also found in equivalent positions of the rat sequence. Nucleas S1 protection mapping revealed the second promoter to be preferentially used in most tissues or in hepatoma cells, and the second poly A addition signal to be the only one functional in all the RNA sources examined. Images PMID:3306601

  11. Exon array data analysis using Affymetrix power tools and R statistical software

    PubMed Central

    2011-01-01

    The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform. PMID:21498550

  12. Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas.

    PubMed

    Murakami, A; Mori, T; Sakai, H; Murakami, M; Yanai, T; Hoshino, Y; Maruo, K

    2011-09-01

    KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.

  13. 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

  14. Classifying nucleic acid sub-sequences as introns or exons using genetic programming

    SciTech Connect

    Handley, S.

    1995-12-31

    An evolutionary computation technique, genetic programming, created programs that classify messenger RNA sequences into one of two classes: (1) the sequence is expressed as (part of) a protein (an exon), or (2) not expressed as protein (an intron).

  15. 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.

  16. Exon array data analysis using Affymetrix power tools and R statistical software.

    PubMed

    Lockstone, Helen E

    2011-11-01

    The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform.

  17. 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.

  18. 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.

  19. 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.

  20. Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins.

    PubMed

    Ramírez-Sánchez, Obed; Pérez-Rodríguez, Paulino; Delaye, Luis; Tiessen, Axel

    2016-12-01

    Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa), average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt)]. Streptophyta have on average only ∼5.7 exons of medium size (∼230nt). Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400nt). Among subcellular compartments, membrane proteins are the largest (∼520aa), whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240aa). Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes.

  1. EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics.

    PubMed

    Arcila, Maria E; Nafa, Khedoudja; Chaft, Jamie E; Rekhtman, Natasha; Lau, Christopher; Reva, Boris A; Zakowski, Maureen F; Kris, Mark G; Ladanyi, Marc

    2013-02-01

    In contrast to other primary epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas, insertions in exon 20 of EGFR have been generally associated with resistance to EGFR-tyrosine kinase inhibitors. Their molecular spectrum, clinicopathologic characteristics, and prevalence are not well established. Tumors harboring EGFR exon 20 insertions were identified through an algorithmic screen of 1,500 lung adenocarcinomas. Cases were first tested for common mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and, if negative, further analyzed for EGFR exon 20 insertions. All samples underwent extended genotyping for other driver mutations in EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, MEK1, and AKT by mass spectrometry; a subset was evaluated for ALK rearrangements. We identified 33 EGFR exon 20 insertion cases [2.2%, 95% confidence interval (CI), 1.6-3.1], all mutually exclusive with mutations in the other genes tested (except PIK3CA). They were more common among never-smokers (P < 0.0001). There was no association with age, sex, race, or stage. Morphologically, tumors were similar to those with common EGFR mutations but with frequent solid histology. Insertions were highly variable in position and size, ranging from 3 to 12 bp, resulting in 13 different insertions, which, by molecular modeling, are predicted to have potentially different effects on erlotinib binding. EGFR exon 20 insertion testing identifies a distinct subset of lung adenocarcinomas, accounting for at least 9% of all EGFR-mutated cases, representing the third most common type of EGFR mutation after exon 19 deletions and L858R. Insertions are structurally heterogeneous with potential implications for response to EGFR inhibitors.

  2. 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

  3. The association of ICAM-1 Exon 6 (E469K) but not of ICAM-1 Exon 4 (G241R) and PECAM-1 Exon 3 (L125V) polymorphisms with the development of differentiation syndrome in acute promyelocytic leukemia.

    PubMed

    Dore, Adriana I; Santana-Lemos, Barbara A A; Coser, Virginia M; Santos, Flávia L S; Dalmazzo, Leandro F; Lima, Ana S G; Jacomo, Rafael H; Elias, Jorge; Falcão, Roberto Passetto; Pereira, Waldir V; Rego, Eduardo M

    2007-11-01

    The use of all trans-retinoic acid (ATRA) is the basis of treatment of acute promyelocytic leukemia (APL) and represents the paradigm of differentiation therapy. In general, ATRA is well-tolerated but may be associated with a potentially lethal side-effect, referred to as retinoic acid or differentiation syndrome (DS). The cellular and molecular mechanisms of DS are poorly understood and involve changes in the adhesive qualities and cytokine secretion of leukemic cells during ATRA-induced differentiation. As leukocyte extravasation is a key event in DS pathogenesis, we analyzed the association between the polymorphisms at Exon 4 (G241R) and Exon 6 (E469K) of ICAM-1 and Exon 3 (L125V) of PECAM-1 genes with DS development in APL patients treated with ATRA and anthracyclines. DS was diagnosed in 23/127 (18.1%) APL patients at an average of 11.5 days after the start of ATRA. All patients presented respiratory distress associated with increased ground-glass opacity in chest radiographies. Other accompanying symptoms were: fever not attributable to infection (65.2%), generalized edema (37.5%), weight gain (37.5%), and impairment of renal function (8.6%). We detected an association between development of DS and the AA genotype at Codon 469 of ICAM-1 (odds ratio of 3.5; 95% confidence interval: 1.2-10.2). Conversely, no significant association was detected between G241R or L125V polymorphisms at Exon 4 of ICAM-1 and Exon 3 of PECAM-1, respectively. Our results suggest that susceptibility to DS in APL patients may be influenced by genetic variation in adhesion molecule loci.

  4. Non-Ambulant Duchenne Patients Theoretically Treatable by Exon 53 Skipping have Severe Phenotype

    PubMed Central

    Servais, Laurent; Montus, Marie; Guiner, Caroline Le; Ben Yaou, Rabah; Annoussamy, Mélanie; Moraux, Amélie; Hogrel, Jean-Yves; Seferian, Andreea M.; Zehrouni, Karima; Le Moing, Anne-Gaëlle; Gidaro, Teresa; Vanhulle, Catherine; Laugel, Vincent; Butoianu, Nina; Cuisset, Jean-Marie; Sabouraud, Pascal; Cances, Claude; Klein, Andrea; Leturcq, France; Moullier, Philippe; Voit, Thomas

    2015-01-01

    Abstract Background: Exon skipping therapy is an emerging approach in Duchenne Muscular Dystrophy (DMD). Antisense oligonucleotides that induce skipping of exon 51, 44, 45, or 53 are currently being evaluated in clinical trials. These trials were designed on the basis of data available in general DMD population. Objectives: Our objective was to compare the clinical and functional statuses of non-ambulant DMD patients theoretically treatable by exon 53 skipping and of DMD patients with other mutations. Methods: We first compared fifteen non-ambulant DMD patients carrying deletions theoretically treatable by exon 53 skipping (DMD-53) with fifteen closely age-matched DMD patients with mutations not treatable by exon 53 skipping (DMD-all-non-53) then with fifteen DMD patients carrying deletions not treatable by exon 53 skipping (DMD-del-non-53). Results: We found that DMD-53 patients had a lower left ventricular ejection fraction, more contractures and they tend to have weaker grips and pinch strengths than other DMD patients. DMD-53 patients lost ambulation significantly younger than other DMD patients. This result was confirmed by comparing ages at loss of ambulation in all non-ambulant DMD patients of the DMD cohort identified in a molecular diagnostic lab. Conclusions: These prospective and retrospective data demonstrate that DMD-53 patients have clinically more severe phenotypes than other DMD patients. PMID:27858743

  5. Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes

    PubMed Central

    Tucker, Tracy; Zahir, Farah R; Griffith, Malachi; Delaney, Allen; Chai, David; Tsang, Erica; Lemyre, Emmanuelle; Dobrzeniecka, Sylvia; Marra, Marco; Eydoux, Patrice; Langlois, Sylvie; Hamdan, Fadi F; Michaud, Jacques L; Friedman, Jan M

    2014-01-01

    Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis. PMID:24253858

  6. Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes.

    PubMed

    Tucker, Tracy; Zahir, Farah R; Griffith, Malachi; Delaney, Allen; Chai, David; Tsang, Erica; Lemyre, Emmanuelle; Dobrzeniecka, Sylvia; Marra, Marco; Eydoux, Patrice; Langlois, Sylvie; Hamdan, Fadi F; Michaud, Jacques L; Friedman, Jan M

    2014-06-01

    Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis.

  7. Global regulatory mechanism underlying the activation of an exon network required for neurogenesis

    PubMed Central

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

    2015-01-01

    SUMMARY The vertebrate and neural-specific 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 non-neural 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

  8. 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

  9. Signal-exon trap: a novel method for the identification of signal sequences from genomic DNA

    PubMed Central

    Péterfy, Miklós; Gyuris, Tibor; Takács, László

    2000-01-01

    We describe a genomic DNA-based signal sequence trap method, signal-exon trap (SET), for the identification of genes encoding secreted and membrane-bound proteins. SET is based on the coupling of an exon trap to the translation of captured exons, which allows screening of the exon-encoded polypeptides for signal peptide function. Since most signal sequences are expected to be located in the 5′-terminal exons of genes, we first demonstrate that trapping of these exons is feasible. To test the applicability of SET for the screening of complex genomic DNA, we evaluated two critical features of the method. Specificity was assessed by the analysis of random genomic DNA and efficiency was demonstrated by screening a 425 kb YAC known to contain the genes of four secretory or membrane-bound proteins. All trapped clones contained a translation initiation signal followed by a hydrophobic stretch of amino acids representing either a known signal peptide, transmembrane domain or novel sequence. Our results suggest that SET is a potentially useful method for the isolation of signal sequence-containing genes and may find application in the discovery of novel members of known secretory gene clusters, as well as in other positional cloning approaches. PMID:10710443

  10. A diverse epigenetic landscape at human exons with implication for expression

    PubMed Central

    Singer, Meromit; Kosti, Idit; Pachter, Lior; Mandel-Gutfreund, Yael

    2015-01-01

    DNA methylation is an important epigenetic marker associated with gene expression regulation in eukaryotes. While promoter methylation is relatively well characterized, the role of intragenic DNA methylation remains unclear. Here, we investigated the relationship of DNA methylation at exons and flanking introns with gene expression and histone modifications generated from a human fibroblast cell-line and primary B cells. Consistent with previous work we found that intragenic methylation is positively correlated with gene expression and that exons are more highly methylated than their neighboring intronic environment. Intriguingly, in this study we identified a unique subset of hypomethylated exons that demonstrate significantly lower methylation levels than their surrounding introns. Furthermore, we observed a negative correlation between exon methylation and the density of the majority of histone modifications. Specifically, we demonstrate that hypo-methylated exons at highly expressed genes are associated with open chromatin and have a characteristic histone code comprised of significantly high levels of histone markings. Overall, our comprehensive analysis of the human exome supports the presence of regulatory hypomethylated exons in protein coding genes. In particular our results reveal a previously unrecognized diverse and complex role of the epigenetic landscape within the gene body. PMID:25765649

  11. BRAF exon 15 mutations in pediatric renal stromal tumors: prevalence in metanephric stromal tumors.

    PubMed

    Marsden, Lily; Jennings, Lawrence J; Gadd, Samantha; Yu, Min; Perlman, Elizabeth J; Cajaiba, Mariana M

    2017-02-01

    Metanephric stromal tumors (MSTs) are rare renal stromal tumors that predominantly affect children. They belong to the metanephric family of tumors, along with metanephric adenofibroma and metanephric adenoma. The previous documentation of BRAF exon 15 mutations in 88% of metanephric adenomas and in isolated cases of metanephric adenofibroma prompted us to investigate the prevalence of these mutations in MSTs and in other pediatric renal stromal tumors. In this study, 17 MSTs, 22 congenital mesoblastic nephromas, and 6 ossifying renal tumors of infancy were selected for BRAF exon 15 testing. Tumor genomic DNA was extracted from formalin-fixed paraffin-embedded tissue, followed by polymerase chain reaction amplification and Sanger dideoxy sequencing with primers flanking the BRAF exon 15 gene. BRAF exon 15 mutations were found in 11 (65%) of the 17 cases of MST, all corresponding to a thymidine-to-adenine substitution at codon 600 (BRAF V600E). All other renal stromal tumors tested were negative for BRAF exon 15 mutations. In conclusion, BRAF V600E mutations are encountered in most MSTs, supporting a link with other metanephric tumors and suggesting a clonal event possibly affecting primordial renal cells. In addition, BRAF V600E mutations have been associated with oncogene-induced senescence in other benign tumors, providing clues to the pathogenesis of metanephric neoplasms in keeping with their overall benign behavior. Our results also suggest a potential diagnostic use for BRAF exon 15 mutations in differentiating MSTs from other pediatric renal stromal tumors, particularly in limited samples.

  12. Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons.

    PubMed

    Partono, S; Lewin, A S

    1990-11-01

    Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.

  13. A multiplexed fragment analysis-based assay for detection of JAK2 exon 12 mutations.

    PubMed

    Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2013-09-01

    Mutations within exon 12 of the JAK2 gene occur in most cases of JAK2 V617F-mutation negative polycythemia vera. Several methods have been developed to identify exon 12 mutations, with both Sanger sequencing and high resolution melting (HRM) being widely used. However, mutations can occur at allelic levels lower than 15%, which may hamper detection by these methods. We developed a novel fragment analysis-based assay capable of detecting nearly all JAK2 exon 12 mutations associated with polycythemia vera down to a sensitivity of 2% mutant allele. Test results were reviewed from a set of 20 reference cases and 1731 consecutive specimens that were referred to our laboratory for testing. Assay performance was compared to sequencing and HRM across a series of 27 specimens with JAK2 exon 12 mutations. Positive cases consisted of 22 with deletion mutations, four with duplications, and one with K539L. Nine cases had mutation levels between 6% and 15% that may not be reliably detected by sequencing or HRM. All cases were easily interpreted in the fragment analysis assay. Sequencing, HRM, and fragment analysis each represent viable platforms for detection of JAK2 exon 12 mutations. Our method performed favorably by providing a simple, robust, and highly sensitive solution for JAK2 exon 12 mutation testing.

  14. NF1 single and multi-exons copy number variations in neurofibromatosis type 1.

    PubMed

    Imbard, Apolline; Pasmant, Eric; Sabbagh, Audrey; Luscan, Armelle; Soares, Magali; Goussard, Philippe; Blanché, Hélène; Laurendeau, Ingrid; Ferkal, Salah; Vidaud, Michel; Pinson, Stéphane; Bellanne-Chantelot, Christine; Vidaud, Dominique; Wolkenstein, Pierre; Parfait, Béatrice

    2015-04-01

    Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

  15. Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells.

    PubMed

    van Deutekom, J C; Bremmer-Bout, M; Janson, A A; Ginjaar, I B; Baas, F; den Dunnen, J T; van Ommen, G J

    2001-07-15

    Due to frame-shifting mutations in the DMD gene that cause dystrophin deficiency, Duchenne muscular dystrophy (DMD) patients suffer from lethal muscle degeneration. In contrast, mutations in the allelic Becker muscular dystrophy (BMD) do not disrupt the translational reading frame, resulting in a less severe phenotype. In this study, we explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, we set up an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in only approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. Our results provide first evidence of highly effective restoration of dystrophin expression from the endogenous gene in DMD patient-derived muscle cells. This strategy may be applicable to not only >65% of DMD mutations, but also many other genetic diseases.

  16. Dantrolene enhances antisense-mediated exon skipping in human and mouse models of Duchenne muscular dystrophy.

    PubMed

    Kendall, Genevieve C; Mokhonova, Ekaterina I; Moran, Miriana; Sejbuk, Natalia E; Wang, Derek W; Silva, Oscar; Wang, Richard T; Martinez, Leonel; Lu, Qi L; Damoiseaux, Robert; Spencer, Melissa J; Nelson, Stanley F; Miceli, M Carrie

    2012-12-12

    Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.

  17. Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster.

    PubMed

    May, Gemma E; Olson, Sara; McManus, C Joel; Graveley, Brenton R

    2011-02-01

    Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.

  18. Isolation of the Male-Specific Transformer Exon as a Method for Immature Specimen Sex Identification in Chrysomya megacephala (Diptera: Calliphoridae).

    PubMed

    Smith, J L; Wells, J D

    2016-12-28

    Being able to efficiently differentiate between male and female individuals in the immature forms of insects allows for investigations into sexually dimorphic patterns of growth rates and gene expression. For species lacking sex-specific morphological characteristics during these periods, alternative methods must be devised. Commonly, isolation of sex determination genes reveals sex-specific band patterns and allows for markers that can be used in insect control. For blow flies, a family that includes flies of medical and forensic importance, sex has previously been identified in some members using the male-specific exon in the transformer gene. This gene is relatively conserved between members of the genera Cochliomyia and Lucilia (Diptera: Calliphoridae), and we isolated a portion of this gene in an additional forensically and medically important blow fly genus using the widespread Chrysomya megacephala (F.). We found a relatively high level of conservation between exons 1 and 2 of transformer and were able to amplify a region containing the male-specific exon in C. megacephala A sex-specific molecular diagnostic test based on the presence of sexually dimorphic PCR product bands showed the expected genotype for adults and intrapuparial period specimens of known sex. The same result could be obtained from single third-instar larval specimens, opening up the possibility to not only determine if development rates are sex dependent, but also to investigate the development of sexually dimorphic traits of interest in C. megacephala.

  19. 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%.

  20. Systematic Dissection of Coding Exons at Single Nucleotide Resolution Supports an Additional Role in Cell-Specific Transcriptional Regulation

    PubMed Central

    Kim, Mee J.; Findlay, Gregory M.; Martin, Beth; Zhao, Jingjing; Bell, Robert J. A.; Smith, Robin P.; Ku, Angel A.; Shendure, Jay; Ahituv, Nadav

    2014-01-01

    In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types. PMID:25340400

  1. RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts.

    PubMed

    Li, Yang I; Sanchez-Pulido, Luis; Haerty, Wilfried; Ponting, Chris P

    2015-01-01

    Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein-protein interactions.

  2. Deciphering the impact of somatic mutations in exon 20 and exon 9 of PIK3CA gene in breast tumors among Indian women through molecular dynamics approach.

    PubMed

    Sudhakar, N; Priya Doss, C George; Thirumal Kumar, D; Chakraborty, Chiranjib; Anand, Kushi; Suresh, M

    2016-01-01

    We examined 25 breast tumor samples for somatic mutations in exon 20 and exon 9 of PIK3CA gene in South Indian population. Genomic DNA was isolated and amplified for PIK3CA gene, followed by direct sequencing of purified polymerase chain reaction products. We identified PI3K3CA mutations in 5 of 25 (20%), including four of the mutations in p.H1047R and one in p.H1047L. Nucleotide base substitution A to G (c.3140A > G) and A to T (c.3140A > T) results in p.H1047R and p.H1047L mutation in exon 20 of PIK3CA gene. We did not observe any mutation in exon 9 of PIK3CA gene. Furthermore, we investigated the effect of mutations on protein structure and function by the combination of sequence and structure-based in silico prediction methods. This determined the underlying relationship between the mutation and its phenotypic effects. Next step, we complemented by molecular dynamics simulation analysis (30 ns) of native and mutant structures that measured the effect of mutation on protein structure. The obtained results support that the application of computational methods helps predict the biological significance of mutations.

  3. 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

  4. Canine and human gastrointestinal stromal tumors display similar mutations in c-KIT exon 11

    PubMed Central

    2010-01-01

    Background Gastrointestinal stromal tumors (GISTs) are common mesenchymal neoplasms in the gastrointestinal tract of humans and dogs. Little is known about the pathogenesis of these tumors. This study evaluated the role of c-KIT in canine GISTs; specifically, we investigated activating mutations in exons 8, 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), all of which have been implicated in human GISTs. Methods Seventeen canine GISTs all confirmed to be positive for KIT immunostaining were studied. Exons 8, 9, 11, 13 and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA, were amplified from DNA isolated from formalin-fixed paraffin-embedded samples. Results Of these seventeen cases, six amplicons of exon 11 of c-KIT showed aberrant bands on gel electrophoresis. Sequencing of these amplicons revealed heterozygous in-frame deletions in six cases. The mutations include two different but overlapping six base pair deletions. Exons 8, 9, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA had no abnormalities detected by electrophoresis and sequencing did not reveal any mutations, other than synonymous single nucleotide polymorphisms (SNPs) found in exon 11 of c-KIT and exons 12 and 14 of PDGFRA. Conclusions The deletion mutations detected in canine GISTs are similar to those previously found in the juxtamembrane domain of c-KIT in canine cutaneous mast cell tumors in our laboratory as well as to those reported in human GISTs. Interestingly, none of the other c-KIT or PDGFRA exons showed any abnormalities in our cases. This finding underlines the critical importance of c-KIT in the pathophysiology of canine GISTs. The expression of KIT and the identification of these activating mutations in c-KIT implicate KIT in the pathogenesis of these tumors. Our results indicate that mutations in c-KIT may be of prognostic significance and that targeting KIT may be a rational approach to treatment of these

  5. Novel frame shift mutations ('A' deletion) observed in exon 9 of Wilms' tumor (WT1) gene in a patient reported with glomerulosclerosis.

    PubMed

    Pasupuleti, Santhosh Kumar; Katari, Venkatesh; Lokanathan, Srikanth; Uppu, Venkateswara Prasad; Thummaginjala, Syama Sundar; Akkamgari, Ram Prasad Reddy; Ayapati, Tyagi; Kottu, Radhika; Potukuchi, Venkata Gurunadha Krishna Sarma

    2014-08-01

    Wilms' tumor-suppressor gene-1 (WT1) is a transcription factor that contains four zinc-finger motifs at the C-terminus and plays a crucial role in kidney and gonad development. We have identified primitive glomeruloid formation using immunohistochemistry in a patient who was clinically diagnosed with a Wilms' tumor. In order to understand the involvement of mutations in the WT1 gene, the genomic DNA was isolated from peripheral blood of the patient (18/F). Exon 9 of the WT1 gene was amplified and sequenced. The obtained sequence was BLAST searched against the transcript variants (TV) of the WT1 gene. An amplified exon 9 sequence of the WT1 gene showing similarity with exon 9 of TV-A, F and exon 10 of TV-B, D and E with a deletion of single nucleotide 'A' causing frame shift in the 4th zinc finger domain of the WT1 protein resulted in Wilms' tumor condition. The deletion position is variable with different transcript variants and they are present at: for TV-A c.1592delA, p.468, for TV-F c.1053delA, p.259, for TV-B c.1643delA, p.485, for TV-D c.1652 delA, p.488, and for TV-E c.1095delA, p.273; all these variations resulted in frame shift mutation. In order to substantiate these results in silico analysis was carried out; the structural superimposition of wild type and mutant WT1 structures showed that the mutated region exhibited a different confirmation with RMSD of 1.759Å. Therefore, these results conclusively explain the mutation in the WT1 gene that leads to structural changes contributing to glomerulosclerosis.

  6. Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

    PubMed Central

    Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O.; Decker, Christian; Preising, Markus N.; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Issa, Peter Charbel; Holz, Frank G.; Baig, Shahid M.; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y.; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S.; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J.

    2013-01-01

    Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even

  7. Breed-specific expression of GR exon 1 mRNA variants and profile of GR promoter CpG methylation in the hippocampus of newborn piglets.

    PubMed

    Sun, Q; Jia, Y; Li, R; Li, X; Yang, X; Zhao, R

    2014-11-01

    Glucocorticoid receptor (GR) transcription is driven by alternative promoters to produce different exon 1 mRNA variants. CpG methylation on GR promoters profoundly affects GR transcription. GR in hippocampus is critical for energy homeostasis and stress responses, yet it remains unclear whether hippocampal expression of GR exon 1 mRNA variants and the methylation status of GR promoters differ between Large White (LW) and Erhualian (EHL) pigs showing distinct metabolic and stress-coping characteristics. EHL pigs had higher hippocampus weight relative to BW (P<0.01), which was associated with higher serum cortisol level compared with LW pigs. Hippocampal expression of brain-derived neurotrophic factor (P<0.05) was significantly higher, while Bax, a pro-apoptotic gene, was significantly lower in EHL pigs (P<0.05). Hippocampal expression of total GR did not differ between breeds, yet GR exon 1 to 11 mRNA was significantly higher (P<0.01) in EHL pigs, which was associated with a trend of increase (P=0.057) in GR protein content. No significant breed difference was detected for the methylation status across the whole region of the proximal GR promoter, while CpG334 and CpG266.267 were differentially methylated, in a reversed manner, between breeds. The methylation status of CpGs 248, 259, 260, 268 and 271 was negatively correlated (P<0.05) with GR exon 1 to 11 mRNA abundance. Our results provide fundamental information on the breed-specific characteristics of GR and its mRNA variants expression and the status of DNA methylation on the proximal GR promoter in the pig hippocampus.

  8. 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.

  9. Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies.

    PubMed

    Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O; Decker, Christian; Preising, Markus N; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Charbel Issa, Peter; Holz, Frank G; Baig, Shahid M; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J

    2013-01-01

    Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even

  10. Current Bacterial Gene Encoding Capsule Biosynthesis Protein CapI Contains Nucleotides Derived from Exonization

    PubMed Central

    Wang, Yong; Tao, Xia-Fang; Su, Zhi-Xi; Liu, A-Ke; Liu, Tian-Lei; Sun, Ling; Yao, Qin; Chen, Ke-Ping; Gu, Xun

    2016-01-01

    Since the proposition of introns-early hypothesis, although many studies have shown that most eukaryotic ancestors possessed intron-rich genomes, evidence of intron existence in genomes of ancestral bacteria has still been absent. While not a single intron has been found in all protein-coding genes of current bacteria, analyses on bacterial genes horizontally transferred into eukaryotes at ancient time may provide evidence of intron existence in bacterial ancestors. In this study, a bacterial gene encoding capsule biosynthesis protein CapI was found in the genome of sea anemone, Nematostella vectensis. This horizontally transferred gene contains a phase 1 intron of 40 base pairs. The nucleotides of this intron have high sequence identity with those encoding amino acids in current bacterial CapI gene, indicating that the intron and the amino acid-coding nucleotides are originated from the same ancestor sequence. Moreover, 5′-splice site of this intron is located in a GT-poor region associated with a closely following AG-rich region, suggesting that deletion mutation at 5′-splice site has been employed to remove this intron and the intron-like amino acid-coding nucleotides in current bacterial CapI gene are derived from exonization. These data suggest that bacterial CapI gene contained intron(s) at ancient time. This is the first report providing the result of sequence analysis to suggest possible existence of spliceosomal introns in ancestral bacterial genes. The methodology employed in this study may be used to identify more such evidence that would aid in settlement of the dispute between introns-early and introns-late theories. PMID:27980385

  11. 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.

  12. 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.

  13. 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

    2016-05-20

    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.

  14. 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.

  15. 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.

  16. Altered Striatal Synaptic Function and Abnormal Behaviour in Shank3 Exon4–9 Deletion Mouse Model of Autism

    PubMed Central

    Jaramillo, Thomas C.; Speed, Haley E.; Xuan, Zhong; Reimers, Jeremy M.; Liu, Shunan; Powell, Craig M.

    2016-01-01

    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 (Shank3e4–9 KO) results in loss of the two highest molecular weight isoforms of Shank3 and a significant reduction in other isoforms. Behaviorally, both Shank3e4–9 heterozygous (HET) and Shank3e4–9 KO mice display increased repetitive grooming, deficits in novel and spatial object recognition learning and memory, and abnormal ultrasonic vocalizations. Shank3e4–9 KO mice also display abnormal social interaction when paired with one another. Analysis of synaptosome fractions from striata of Shank3e4–9 KO mice reveals decreased Homer1b/c, GluA2, and GluA3 expression. Both Shank3e4–9 HET and KO demonstrated a significant reduction in NMDA/AMPA ratio at excitatory synapses onto striatal medium spiny neurons. Furthermore, Shank3e4–9 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 Shank3e4–9 mutant mouse model of autism. PMID:26559786

  17. hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2).

    PubMed

    Tang, Xiaoyu; Kane, Vanessa D; Morré, Dorothy M; Morré, D James

    2011-11-01

    HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.

  18. 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

  19. Cellular Localization and Processing of Primary Transcripts of Exonic MicroRNAs

    PubMed Central

    Slezak-Prochazka, Izabella; Kluiver, Joost; de Jong, Debora; Kortman, Gertrud; Halsema, Nancy; Poppema, Sibrand; Kroesen, Bart-Jan; van den Berg, Anke

    2013-01-01

    Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC, <1 for pri-miR-146a and variable for pri-miR-22. Endogenous unspliced transcripts were located almost exclusively in the nucleus and thus available for miRNA processing for all three miRNAs. Endogenous spliced pri-miRNA transcripts were present both in the nucleus and in the cytoplasm and thus only partly available for miRNA processing. Overexpression of constructs containing the 5’ upstream exonic or intronic sequence flanking pre-miR-155 resulted in strongly enhanced miR-155 levels, indicating that the flanking sequence does not affect processing efficiency. Exogenously overexpressed full-length spliced BIC transcripts were present both in the nucleus and in the cytoplasm, were bound by the Microprocessor complex and resulted in enhanced miR-155 levels. We conclude that both unspliced and spliced transcripts of exonic miRNAs can be used for pre-miRNA cleavage. Splicing and cytoplasmic transport of spliced transcripts may present a mechanism to regulate levels of exonic microRNAs. PMID:24073292

  20. Lack of exon 10 in the murine tau gene results in mild sensorimotor defects with aging

    PubMed Central

    2013-01-01

    Background Complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease. Results Gene-targeting was used to delete exon 10 in murine tau on both alleles (E10−/−) to study its functional role. Moreover, mice devoid of exon 10 (E10+/−) on one allele were generated to investigate the effects of 1:1 balanced expression of 4R-/3R-tau protein, since equal amounts of 4R-/3R-tau protein are synthesized in human brain. Middle-aged E10−/− mice displayed sensorimotor disturbances in the rotarod when compared to age-matched E10+/− and wild-type mice, and their muscular grip strength was less than that of E10+/− mice. The performance of E10+/− mice and wild-type mice (E10+/+) was similar in sensorimotor tests. Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found. Conclusion Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. Presumably related microtubule-stabilizing genes rescue other functions. PMID:24261309

  1. Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene

    SciTech Connect

    Zhi Chen; Kellems, R.E.; Innis, J.W. ); Sun, Minghua; Wright, D.A. )

    1991-12-01

    The authors have previously demonstrated that a transcriptional arrest site exists in exon 1 of the human adenosine deaminase (ADA) gene and that this site may play a role in ADA gene expression. Sequences involved in this process are not known precisely. To further define the template requirements for transcriptional arrest within exon 1 of the human ADA gene, various ADA templates were constructed and their abilities to confer transcriptional arrest were determined following injection into Xenopus oocytes. The exon 1 transcriptional arrest signal functioned downstream of several RNA polymerase II promoters and an RNA polymerase II promoter, implying that the transcriptional arrest site in exon 1 of the ADA gene is promoter independent. They identified a 43-bp DNA fragment which functions as a transcriptional arrest signal. Additional studies showed that the transcriptional arrest site functioned only in the naturally occurring orientation. Therefore, they have identified a 43-bp DNA fragment which functions as a transcriptional arrest signal in an orientation-dependent and promoter-independent manner. On the basis of the authors findings, they hypothesize that tissue-specific expression of the ADA gene is governed by factors that function as antiterminators to promote transcriptional readthrough of the exon 1 transcriptional arrest site.

  2. A computational and experimental approach toward a priori identification of alternatively spliced exons

    PubMed Central

    PHILIPPS, DANA L.; PARK, JUNG W.; GRAVELEY, BRENTON R.

    2004-01-01

    Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data. PMID:15525709

  3. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core.

    PubMed

    Hoop, Cody L; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M; Boatz, Jennifer C; Mandal, Abhishek; Lewandowski, Józef R; Wetzel, Ronald; van der Wel, Patrick C A

    2016-02-09

    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.

  4. 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

  5. Noncoder: a web interface for exon array-based detection of long non-coding RNAs

    PubMed Central

    Gellert, Pascal; Ponomareva, Yuliya; Braun, Thomas; Uchida, Shizuka

    2013-01-01

    Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called ‘noncoder’. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de. PMID:23012263

  6. TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy.

    PubMed

    Singh, Natalia N; Seo, Joonbae; Ottesen, Eric W; Shishimorova, Maria; Bhattacharya, Dhruva; Singh, Ravindra N

    2011-03-01

    Prevention of skipping of exon 7 during pre-mRNA splicing of Survival Motor Neuron 2 (SMN2) holds the promise for cure of spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Here, we report T-cell-restricted intracellular antigen 1 (TIA1) and TIA1-related (TIAR) proteins as intron-associated positive regulators of SMN2 exon 7 splicing. We show that TIA1/TIAR stimulate exon recognition in an entirely novel context in which intronic U-rich motifs are separated from the 5' splice site by overlapping inhibitory elements. TIA1 and TIAR are modular proteins with three N-terminal RNA recognition motifs (RRMs) and a C-terminal glutamine-rich (Q-rich) domain. Our results reveal that any one RRM in combination with a Q domain is necessary and sufficient for TIA1-associated regulation of SMN2 exon 7 splicing in vivo. We also show that increased expression of TIA1 counteracts the inhibitory effect of polypyrimidine tract binding protein, a ubiquitously expressed factor recently implicated in regulation of SMN exon 7 splicing. Our findings expand the scope of TIA1/TIAR in genome-wide regulation of alternative splicing under normal and pathological conditions.

  7. A bioinformatics analysis of alternative exon usage in human genes coding for extracellular matrix proteins.

    PubMed

    Sakabe, Noboru Jo; Vibranovski, Maria Dulcetti; de Souza, Sandro José

    2004-12-30

    Alternative splicing increases protein diversity through the generation of different mRNA molecules from the same gene. Although alternative splicing seems to be a widespread phenomenon in the human transcriptome, it is possible that different subgroups of genes present different patterns, related to their biological roles. Analysis of a subgroup may enhance common features of its members that would otherwise disappear amidst a heterogeneous population. Extracellular matrix (ECM) proteins are a good set for such analyses since they are structurally and functionally related. This family of proteins is involved in a large variety of functions, probably achieved by the combinatorial use of protein domains through exon shuffling events. To determine if ECM genes have a different pattern of alternative splicing, we compared clusters of expressed sequences of ECM to all other genes regarding features related to the most frequent type of alternative splicing, alternative exon usage (AEU), such as: the number of alternative exon-intron structures per cluster, the number of AEU events per exon-intron structure, the number of exons per event, among others. Although we did not find many differences between the two sets, we observed a higher frequency of AEU events involving entire protein domains in the ECM set, a feature that could be associated with their multi-domain nature. As other subgroups or even the ECM set in different tissues could present distinct patterns of AEU, it may be premature to conclude that alternative splicing is homogeneous among groups of related genes.

  8. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    PubMed

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients.

  9. Targeted skipping of human dystrophin exons in transgenic mouse model systemically for antisense drug development.

    PubMed

    Wu, Bo; Benrashid, Ehsan; Lu, Peijuan; Cloer, Caryn; Zillmer, Allen; Shaban, Mona; Lu, Qi Long

    2011-01-01

    Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD mouse, a transgenic model carrying the full-length human dystrophin gene, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.

  10. 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

  11. KIT exon 11 deletion-inversions represent complex mutations in gastrointestinal stromal tumors.

    PubMed

    Lasota, Jerzy; Miettinen, Markku

    2007-05-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. KIT expression and mutational KIT activation have been documented in a majority of GISTs. Most mutations have been found in KIT juxtamembrane domain encoded by exon 11. Recently, we have identified three, complex KIT exon 11 mutations previously unreported in GISTs. These mutations consisted of several nucleotide deletions accompanied by insertions of inverted complementary DNA strand sequences. All three mutations were found in the 5' part of KIT exon 11. At the protein level, these mutations lead to the same end result: in-frame loss and insertion of a number of amino acids and could be considered examples of deletion-insertion. Although proper description of these mutations at the genomic level is a complex task and requires an individual approach, the uniform name deletion-inversion is suggested for this type of mutation, based on the present study. The frequency of deletion-inversions among KIT exon 11 mutant GISTs was estimated to be <0.5%, based on evaluation of 700 KIT exon 11 mutants. Molecular events leading to formation of deletion-inversions remain elusive and should be studied further.

  12. High sequence similarity within ras exons 1 and 2 in different mammalian species and phylogenetic divergence of the ras gene family.

    PubMed

    Watzinger, F; Mayr, B; Haring, E; Lion, T

    1998-03-01

    We have determined the canine and feline N-, K-, and H-ras gene sequences from position +23 to +270 covering exons I and II which contain the mutational hot spot codons 12, 13, and 61. The results were used to assess the degree of similarity between ras gene DNA regions containing the critical domains affected in neoplastic disorders in different mammalian species. The comparative analyses performed included human, canine, feline, murine, rattine, and, whenever possible, bovine, leporine (rabbit), porcelline (guinea pig), and mesocricetine (hamster) ras gene sequences within the region of interest. Comparison of feline and canine nucleotide sequences with the corresponding regions in human DNA revealed a sequence similarity greater than 85% to the human sequence. Contemporaneous analysis of previously published ras DNA sequences from other mammalian species showed a similar degree of homology to human DNA. Most nucleotide differences observed represented synonymous changes without effect on the amino acid sequence of the respective proteins. For assessment of the phylogenetic evolution of ras gene family, a maximum parsimony dendrogram based on multiple sequence alignment of the common region of exons I and II in the N-, K-, and H-ras genes was constructed. Interestingly, a higher substitution rate among the H-ras genes became apparent, indicating accelerated sequence evolution within this particular clade. The most parsimonious tree clearly shows that the duplications giving rise to the three ras genes must have occurred before the mammalian radiation.

  13. 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

  14. Immunochemical detection of proteins related to the human c-myc exon 1.

    PubMed Central

    Gazin, C; Rigolet, M; Briand, J P; Van Regenmortel, M H; Galibert, F

    1986-01-01

    Published sequence data of the human c-myc gene indicate the presence of a coding capacity for a polypeptide of 188 residues within the first exon. Using antibodies raised against five synthetic peptides corresponding to different non-over-lapping parts of this polypeptide, two proteins of 32 kd and 58 kd antigenically related to the synthetic peptides have been detected in extracts of human cells. The confidence of this detection has been reinforced by showing that epitopes corresponding to different peptides were indeed located on the same molecule and that the 58 kd protein appears to be a dimeric form of the 32 kd protein. That these proteins originate from the first exon was indicated by: hybrid-arrested translation experiments followed by immunodetection of the translation products; in vitro translation of messenger RNA derived from cloned exon 1 by SP6 polymerase transcription. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2430795

  15. Exon 32 Skipping of Dysferlin Rescues Membrane Repair in Patients’ Cells

    PubMed Central

    Barthélémy, Florian; Blouin, Cédric; Wein, Nicolas; Mouly, Vincent; Courrier, Sébastien; Dionnet, Eugénie; Kergourlay, Virginie; Mathieu, Yves; Garcia, Luis; Butler-Browne, Gillian; Lamaze, Christophe; Lévy, Nicolas; Krahn, Martin; Bartoli, Marc

    2015-01-01

    Abstract Dysferlinopathies are a family of disabling muscular dystrophies with LGMD2B and Miyoshi myopathy as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for a subset of patients with dysferlinopathies. Such an approach aims to rescue functional proteins when targeting modular proteins and specific tissues. We sought to evaluate the dysferlin functional recovery following exon 32 skipping in the cells of affected patients. Exon skipping efficacy was characterized at several levels by use of in vitro myotube formation assays and quantitative membrane repair and recovery tests. Data obtained from these assessments confirmed that dysferlin function is rescued by quasi-dysferlin expression in treated patient cells, supporting the case for a therapeutic antisense-based trial in a subset of dysferlin-deficient patients. PMID:27858744

  16. Genetic characterization of MHC class II DQB exon 2 variants in gayal (Bos frontalis).

    PubMed

    Sun, Yongke; Xi, Dongmei; Li, Guozhi; Hao, Tiantian; Chen, Yuhan; Yang, Yuai

    2014-09-03

    In the present study, exon 2 of major histocompatibility complex (MHC) class II DQB gene from 39 gayals (Bos frontalis) was isolated, characterized and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 36 DQB exon 2 variants among 39 gayals. These variants exhibited a high degree of nucleotide and amino acid substitutions with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (dS) and non-synonymous (dN) substitution. The gayals were observed to be under strong balancing selection in the DQB exon 2 PBS (dN = 0.094, P = 0.001). It appears that this variability among gayals could confer the ability to mount immune responses to a wide variety of peptides or pathogens.

  17. Genetic characterization of MHC class II DQB exon 2 variants in gayal (Bos frontalis)

    PubMed Central

    Sun, Yongke; Xi, Dongmei; Li, Guozhi; Hao, Tiantian; Chen, Yuhan; Yang, Yuai

    2014-01-01

    In the present study, exon 2 of major histocompatibility complex (MHC) class II DQB gene from 39 gayals (Bos frontalis) was isolated, characterized and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 36 DQB exon 2 variants among 39 gayals. These variants exhibited a high degree of nucleotide and amino acid substitutions with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (d S) and non-synonymous (d N) substitution. The gayals were observed to be under strong balancing selection in the DQB exon 2 PBS (d N = 0.094, P = 0.001). It appears that this variability among gayals could confer the ability to mount immune responses to a wide variety of peptides or pathogens. PMID:26019566

  18. 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.

  19. Molecular Genetic Analysis of a Suprasellar Immature Teratoma : Mutation of Exon 4 P53 Gene

    PubMed Central

    Udin, Nujaimin; Ahmad, Ku Asmarina Ku; Ahmad, Farizan; Omar, Effat; Aziz, Mohd Ezanee; Kumar, Raj; Abdullah, Jafri Malin

    2008-01-01

    We described an intracranial immature teratoma in a 13 year old Malay boy who presented with history of chronic headache and blurring of vision. Physical findings revealed bilateral papilloedema but no other localizing sign. A Magnetic Resonance Imaging of the brain revealed a suprasellar well defined lobulated midline heterogenous mass which was intraoperatively described as mainly solid tumour with multiple small cystic component filled with yellowish jelly like material. Histopathological finding confirmed the case as immature teratoma. Molecular genetic analysis of p53 and p27 genes revealed substitution of nucleotide G to C at location nucleotide 12139, exon 4 of gene p53. No alteration was detected at exon 5–6 and 8 of p53 gene and exon 1 and 2 of p27 gene. This is the first case report of an intracranial immature teratoma with genetic mutation occuring in a Malay boy. PMID:22589625

  20. Compartmentalization of PDGF on extracellular binding sites dependent on exon-6-encoded sequences

    PubMed Central

    1992-01-01

    The PDGFs are a family of molecules assembled as disulfide-bonded homo- and heterodimers from two distinct but highly homologous polypeptide chains (PDGF-A and PDGF-B). Two PDGF A-chain transcripts, which arise from alternative usage of the 69-bp exon 6 and exon 7, give rise to two forms of PDGF-A. In spite of the conservation of two PDGF A-chain forms over at least 350 million years, no differences in their biological activities have been identified. We have investigated the activity of the sequence encoded by the alternatively spliced exon 6 of the PDGF A- chain (peptide AL). Addition of peptide AL at 10(-5)-10(-9) M to cultured endothelium and smooth muscle induced a dose-dependent, 3-20- fold increase in PDGF in conditioned media within 30 min. Peptide AL had no detectable effect on A- or B-chain transcript levels, and decrease in culture temperature did not prevent rapid release of PDGF. In human umbilical vein endothelial cells treated with peptide AL, the PDGF release was principally PDGF-BB, while in smooth muscle cells it was primarily PDGF-AA. The capacity to induce release of PDGF is shared by the homologous peptide encoded by exon 6 of the B-chain of PDGF. Binding studies and cross-linking analysis are consistent with a charge- based association of exon 6 sequences with membrane- and matrix- associated heparan-sulfate proteoglycans. We hypothesize that translation of exon 6 of the A- or B-chain of PDGF results in compartmentalization of these forms of PDGF with HS-PG, whereas forms lacking this sequence would be soluble and diffuse. PMID:1309814

  1. Viral diversity and diversification of major non-structural genes vif, vpr, vpu, tat exon 1 and rev exon 1 during primary HIV-1 subtype C infection.

    PubMed

    Rossenkhan, Raabya; Novitsky, Vladimir; Sebunya, Theresa K; Musonda, Rosemary; Gashe, Berhanu A; Essex, M

    2012-01-01

    To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; -0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.

  2. Welcome to the splice age: antisense oligonucleotide–mediated exon skipping gains wider applicability

    PubMed Central

    McNally, Elizabeth M.; Wyatt, Eugene J.

    2016-01-01

    Exon skipping uses antisense oligonucleotides (ASOs) to alter transcript splicing for the purpose of rescuing or modulating protein expression. In this issue of the JCI, Lee and colleagues developed and evaluated an ASO-dependent method for treating certain molecularly defined diseases associated with alterations in lamin A/C (LMNA) splicing. Exon skipping by ASOs is gaining traction as a therapeutic strategy, and the use of ASOs is now being applied to bypass mutations and generate modified but functional proteins for an array of genetic disorders. PMID:26999602

  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. Expression and new exon mutations of the human Beta defensins and their association on colon cancer development.

    PubMed

    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.

  5. [Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans].

    PubMed

    Pepe, Carolina M; Saraco, Nora I; Baquedano, María Sonia; Guercio, Gabriela; Vaiani, Elisa; Berensztein, Esperanza; Rivarola, Marco A; Belgorosky, Alicia

    2007-01-01

    P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

  6. Novel and known MYOC exon 3 mutations in an admixed Peruvian primary open-angle glaucoma population

    PubMed Central

    Mendoza-Reinoso, Veronica; Guevara-Fujita, Maria L.; Fernández, Silvia; Vargas, Enrique; Castillo-Herrera, Wilder; Perez-Grossmann, Rodolfo; Lizaraso-Caparó, Frank; Richards, Julia E.; Fujita, Ricardo

    2012-01-01

    Purpose The aim of this study was to characterize a representative sample of the Peruvian population suffering open-angle glaucoma (OAG) with respect to the myocilin gene (MYOC) mutations, glaucoma phenotype, and ancestry for future glaucoma risk assessment. Methods DNA samples from 414 unrelated Peruvian subjects, including 205 open-angle glaucoma cases (10 juvenile glaucoma [JOAG], 19 normal-tension glaucoma [NTG], and 176 POAG) and 209 randomly sampled controls, were screened for nucleotide changes in MYOC exon 3 by conformational sensitive gel electrophoresis (CSGE) and mutation screening. Results We identified a probable causative novel MYOC missense mutation, Gly326Ser, in one POAG case and found a consistent genotype-phenotype correlation in eight of his relatives. We also found the known causative MYOC mutation Trp286Arg in one JOAG case and one POAG case. A known causative single base MYOC deletion, T1357, was found in one POAG case. Two previously reported silent polymorphisms, Thr325Thr and Tyr347Tyr, were found in both the case and the control populations. A novel missense variant, Met476Arg, was identified in two unrelated controls. Conclusions The screening of exon 3 of MYOC in a representative sample of 205 independent POAG patients from Peru and 209 matched controls identified novel and previously reported mutations (both pathogenic and nonpathogenic) from other global regions. These results reflect the complex admixture of Amerindian and Old World ancestry in urban populations of Latin America, in general, and in Peru, in particular. It will be important to gather information about the ancestral origin of MYOC and other POAG gene mutations to develop screening panels and risk assessment for POAG in Peru. PMID:22879734

  7. Control of Human PLP1 Expression Through Transcriptional Regulatory Elements and Alternatively Spliced Exons in Intron 1

    PubMed Central

    Hamdan, Hamdan; Kockara, Neriman T.; Jolly, Lee Ann; Haun, Shirley

    2015-01-01

    *These authors contributed equally to this work.Although the myelin proteolipid protein gene (PLP1) encodes the most abundant protein in central nervous system (CNS) myelin, not much is known about the mechanisms that govern expression of the human gene (hPLP1). Much more is known about the processes that regulate Plp1 gene expression in rodents. From studies with Plp1-lacZ transgenic mice, it was determined that the first intron of mouse Plp1 (mPlp1) is required to attain high levels of expression in brain, concurrent with the active myelination period. Other studies have suggested that within mPlp1 intron 1 (>8 kb) lie several regions with enhancer-like activity. To test whether these sequences (and possibly others) in hPLP1 intron 1 are functional, deletion-transfection analysis was performed with hPLP1-lacZ constructs that contain various portions of the intron, or lack it altogether. Results presented here demonstrate the importance of hPLP1 intron 1 in achieving maximal levels of expression in the immortalized oligodendroglial cell line, Oli-neu. Deletion analysis indicates that the intron contains multiple positive regulatory elements which are active in Oli-neu cells. Some of these elements appear to be functionally conserved between human and mouse, while others are not. Furthermore, our studies demonstrate that multiple splice variants can be formed due to inclusion of extra (supplementary) exons from what is classically thought of as hPLP1 intron 1. Thus, splicing of these novel exons (which are not recognized as such in mPlp1 due to lack of conserved splice sites) must utilize factors common to both human and mouse since Oli-neu cells are of mouse origin. PMID:25694552

  8. A modified group I intron can function as both a ribozyme and a 5' exon in a trans-exon ligation reaction.

    PubMed

    Tasiouka, K I; Burke, J M

    1994-06-24

    Here, we show that a single RNA molecule derived from a group-I intron can provide the catalytic activity, the substrate recognition domain and the attacking nucleophile in a reaction that mimics the exon ligation step of splicing. To accomplish this reaction, we have linked a 5' exon sequence to the 3' end of an attenuated form of the self-splicing Tetrahymena rRNA intron. The ribozyme (I-E1) attacks an oligoribonucleotide analog of the 3' splice site (I'-E2) to generate a product containing ligated exons (I-E1-E2) and a small intron fragment (I'). Two modified introns were constructed and tested for activity. A construct designed to interact with the 3' splice site through intermolecular P9.0 and P10 helices was found to be inactive due to failure to form a stable ribozyme-substrate complex. A second modified intron and substrate combination was engineered, in which the complex was further stabilized by an intermolecular P9.2 helix. In this case, stable complexes and reaction products were identified. The reaction efficiency was low compared to splicing of the unmodified intron-containing precursor, and will be optimized in future experiments. Following optimization, we believe that this system may be exploited to examine the functional consequences of a wide variety of 3' splice-site modifications, and may provide the basis for development of highly selective trans-acting ribozymes.

  9. Genome-Wide Analyses of Exonic Copy Number Variants in a Family-Based Study Point to Novel Autism Susceptibility Genes

    PubMed Central

    Glessner, Joseph T.; Herman, Edward I.; Sonnenblick, Lisa I.; Alvarez Retuerto, Ana I.; Imielinski, Marcin; Hadley, Dexter; Bradfield, Jonathan P.; Kim, Cecilia; Gidaya, Nicole B.; Lindquist, Ingrid; Hutman, Ted; Sigman, Marian; Kustanovich, Vlad; Lajonchere, Clara M.; Singleton, Andrew; Kim, Junhyong; Wassink, Thomas H.; McMahon, William M.; Owley, Thomas; Sweeney, John A.; Coon, Hilary; Nurnberger, John I.; Li, Mingyao; Cantor, Rita M.; Minshew, Nancy J.; Sutcliffe, James S.; Cook, Edwin H.; Dawson, Geraldine; Buxbaum, Joseph D.; Grant, Struan F. A.; Schellenberg, Gerard D.; Geschwind, Daniel H.; Hakonarson, Hakon

    2009-01-01

    The genetics underlying the autism spectrum disorders (ASDs) is complex and remains poorly understood. Previous work has demonstrated an important role for structural variation in a subset of cases, but has lacked the resolution necessary to move beyond detection of large regions of potential interest to identification of individual genes. To pinpoint genes likely to contribute to ASD etiology, we performed high density genotyping in 912 multiplex families from the Autism Genetics Resource Exchange (AGRE) collection and contrasted results to those obtained for 1,488 healthy controls. Through prioritization of exonic deletions (eDels), exonic duplications (eDups), and whole gene duplication events (gDups), we identified more than 150 loci harboring rare variants in multiple unrelated probands, but no controls. Importantly, 27 of these were confirmed on examination of an independent replication cohort comprised of 859 cases and an additional 1,051 controls. Rare variants at known loci, including exonic deletions at NRXN1 and whole gene duplications encompassing UBE3A and several other genes in the 15q11–q13 region, were observed in the course of these analyses. Strong support was likewise observed for previously unreported genes such as BZRAP1, an adaptor molecule known to regulate synaptic transmission, with eDels or eDups observed in twelve unrelated cases but no controls (p = 2.3×10−5). Less is known about MDGA2, likewise observed to be case-specific (p = 1.3×10−4). But, it is notable that the encoded protein shows an unexpectedly high similarity to Contactin 4 (BLAST E-value = 3×10−39), which has also been linked to disease. That hundreds of distinct rare variants were each seen only once further highlights complexity in the ASDs and points to the continued need for larger cohorts. PMID:19557195

  10. Polymorphism in the Alternative Donor Site of the Cryptic Exon of LHCGR: Functional Consequences and Associations with Testosterone Level

    PubMed Central

    Liu, Wei; Han, Bing; Zhu, Wenjiao; Cheng, Tong; Fan, Mengxia; Wu, Jiajun; Yang, Ying; Zhu, Hui; Si, Jiqiang; Lyu, Qifeng; Chai, Weiran; Zhao, Shuangxia; Song, Huaidong; Kuang, Yanping; Qiao, Jie

    2017-01-01

    Selective splicing is a feature of luteinizing hormone receptor (LHCGR). A cryptic exon (LHCGR-exon 6A) was found to be derived from alternative splicing in intron 6 of the LHCGR gene, which including two transcripts LHCGR-exon 6A-long and LHCGR-exon 6A-short. We addressed the functional consequences of SNP rs68073206, located at the +5 position of an alternative 5′ splice donor site, and observed its association with male infertility in the subjects with azoospermia, oligoasthenozoospermia and normozoospermia. The translation product of splicing variant LHCGR-exon 6A was expressed in the cytoplasm and exhibited no affinity with [125I]-hCG. No dominant negative effect was observed in cells co-expressed with LHCGR-exon 6A and wild-type LHCGR. The long transcript (LHCGR-exon 6A-long) was significantly elevated in the granulosa cells with G/G genotypes, which could be reproduced in vitro by mini-gene construct transfection. Genotyping analysis showed no association between rs68073206 and male infertility. However, this polymorphism was significantly associated with testosterone levels in normozoospermic subjects (n = 210). In conclusion, SNP rs68073206 in the splicing site of the cryptic exon 6A of the LHCGR gene affect the splicing pattern in the gene, which may play a role in the modulation of the LHCGR sensitivity in the gonads. PMID:28367994

  11. Involvement of exon 11-associated variants of the mu opioid receptor MOR-1 in heroin, but not morphine, actions.

    PubMed

    Pan, Ying-Xian; Xu, Jin; Xu, Mingming; Rossi, Grace C; Matulonis, Joshua E; Pasternak, Gavril W

    2009-03-24

    Heroin remains a major drug of abuse and is preferred by addicts over morphine. Like morphine, heroin has high affinity and selectivity for mu-receptors, but its residual analgesia in exon 1 MOR-1 knockout mice that do not respond to morphine suggests a different mechanism of action. MOR-1 splice variants lacking exon 1 have been observed in mice, humans, and rats, raising the possibility that they might be responsible for the residual heroin and morphine-6beta-glucuronide (M6G) analgesia in the exon 1 knockout mice. To test this possibility, we disrupted exon 11 of MOR-1, which eliminates all of the variants that do not contain exon 1. Morphine and methadone analgesia in the exon 11 knockout mouse was normal, but the analgesic actions of heroin, M6G, and fentanyl were markedly diminished in the radiant heat tail-flick and hot-plate assays. Similarly, the ability of M6G to inhibit gastrointestinal transit was greatly diminished in these exon 11 knockout mice, whereas the ability of morphine was unchanged. These findings identify receptors selectively involved with heroin and M6G actions and confirm the relevance of the exon 11-associated variants and raise important issues regarding the importance of atypical truncated G-protein-coupled receptors.

  12. 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.

  13. Oxidative stress controls the choice of alternative last exons via a Brahma-BRCA1-CstF pathway.

    PubMed

    Fontana, Gabriele A; Rigamonti, Aurora; Lenzken, Silvia C; Filosa, Giuseppe; Alvarez, Reinaldo; Calogero, Raffaele; Bianchi, Marco E; Barabino, Silvia M L

    2017-01-25

    Alternative splicing of terminal exons increases transcript and protein diversity. How physiological and pathological stimuli regulate the choice between alternative terminal exons is, however, largely unknown. Here, we show that Brahma (BRM), the ATPase subunit of the hSWI/SNF chromatin-remodeling complex interacts with BRCA1/BARD1, which ubiquitinates the 50 kDa subunit of the 3' end processing factor CstF. This results in the inhibition of transcript cleavage at the proximal poly(A) site and a shift towards inclusion of the distal terminal exon. Upon oxidative stress, BRM is depleted, cleavage inhibition is released, and inclusion of the proximal last exon is favoored. Our findings elucidate a novel regulatory mechanism, distinct from the modulation of transcription elongation by BRM that controls alternative splicing of internal exons.

  14. Oxidative stress controls the choice of alternative last exons via a Brahma–BRCA1–CstF pathway

    PubMed Central

    Fontana, Gabriele A.; Rigamonti, Aurora; Lenzken, Silvia C.; Filosa, Giuseppe; Alvarez, Reinaldo; Calogero, Raffaele; Bianchi, Marco E.; Barabino, Silvia M.L.

    2017-01-01

    Alternative splicing of terminal exons increases transcript and protein diversity. How physiological and pathological stimuli regulate the choice between alternative terminal exons is, however, largely unknown. Here, we show that Brahma (BRM), the ATPase subunit of the hSWI/SNF chromatin-remodeling complex interacts with BRCA1/BARD1, which ubiquitinates the 50 kDa subunit of the 3′ end processing factor CstF. This results in the inhibition of transcript cleavage at the proximal poly(A) site and a shift towards inclusion of the distal terminal exon. Upon oxidative stress, BRM is depleted, cleavage inhibition is released, and inclusion of the proximal last exon is favoored. Our findings elucidate a novel regulatory mechanism, distinct from the modulation of transcription elongation by BRM that controls alternative splicing of internal exons. PMID:27591253

  15. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

    PubMed Central

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S.; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  16. Exonic microdeletions of the gephyrin gene impair GABAergic synaptic inhibition in patients with idiopathic generalized epilepsy.

    PubMed

    Dejanovic, Borislav; Lal, Dennis; Catarino, Claudia B; Arjune, Sita; Belaidi, Abdel A; Trucks, Holger; Vollmar, Christian; Surges, Rainer; Kunz, Wolfram S; Motameny, Susanne; Altmüller, Janine; Köhler, Anna; Neubauer, Bernd A; Epicure Consortium; Nürnberg, Peter; Noachtar, Soheyl; Schwarz, Günter; Sander, Thomas

    2014-07-01

    Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and γ-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5-9 (Δ5-9) and 2-3 (Δ2-3), both affecting the gephyrin G-domain. Molecular characterization of the GPHN Δ5-9 variant demonstrated that it perturbs the clustering of regular gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of γ2-subunit containing GABAARs. GPHN Δ2-3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission.

  17. Defining the ends of Parkin exon 4 deletions in two different families with Parkinson's disease.

    PubMed

    Clarimon, Jordi; Johnson, Janel; Dogu, Okan; Horta, Wagner; Khan, Naheed; Lees, Andrew J; Hardy, John; Singleton, Andrew

    2005-02-05

    Autosomal recessive juvenile parkinsonism (AR-JP, PARK2) is characterized by an early onset parkinsonism, often presenting with dystonia as an early feature. Mutations in Parkin are a relatively common cause of AR-JP and are estimated to be present in approximately 30% of familial young onset Parkinson disease (PD) [Abbas et al. (1999); Hum Mol Genet 8:567-574]. These mutations include exon rearrangements (deletions and duplications), point mutations, and small deletions. Similar genomic mutations have been described in unrelated patients, thereby indicating independent mutational events or ancient founder effects. We have identified homozygous deletion mutations of exon 4 in Parkin in two unrelated families, one from Brazil and the other from Turkey [Dogu et al. (2004); Mov Dis 9:812-816; Khan et al., Mov Dis, in press]. We have performed molecular analysis of the deletion breakpoints and this data indicates these mutations originated independently. We present here data demonstrating that the mutation responsible for disease in the Brazilian kindred consists of two separate deletions (1,069 and 1,750 bp) surrounding and including exon 4. The deletion removing parkin exon 4 identified in the Turkish family extended 156,203 bp. In addition to demonstrating that disease in these families is not caused by a single founder mutation, these data show that there is no common fragile site between these mutational events.

  18. 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.

  19. 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

  20. 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...

  1. Tandemly repeated exons encode 81-base repeats in multiple, developmentally regulated Schistosoma mansoni transcripts.

    PubMed Central

    Davis, R E; Davis, A H; Carroll, S M; Rajkovic, A; Rottman, F M

    1988-01-01

    The adult Schistosoma mansoni cDNA clone 10-3 encodes an antigen that is recognized by sera from infected humans. We characterized multiple developmentally regulated transcripts homologous to the 10-3 cDNA and portions of the complex genomic loci encoding those transcripts. Transcripts of approximately 950, 870, and 780 nucleotides were expressed in adults, whereas only the 780-nucleotide transcript was observed in the larval stage. These transcripts were highly similar, containing variable numbers of identical direct tandem repeats of 81 bases. Although the sequence of the repeating elements and sequences 3' to them were identical in all the transcripts, sequences 5' of the repeating elements exhibited variations, including a 27-base insertion, alternative start sites for transcription, and alternate 5' exon usage. These transcripts appeared to be derived in part by the developmentally controlled alternative splicing of small exons and the use of alternative transcription initiation sites from the one or two complex loci of at least 40 kilobase pairs. Each 81-base repeat in the transcripts was encoded by three dispersed 27-base-pair exons. These 27-base-pair exons were contained within highly conserved, reiterated 3-kilobase-pair genomic tandem arrays. Images PMID:3211127

  2. A novel point mutation in exon 20 of EGFR showed sensitivity to erlotinib.

    PubMed

    Xing, Kailin; Zhou, Xiaoyan; Zhao, Xinmin; Sun, Si; Luo, Zhiguo; Wang, Huijie; Yu, Hui; Wang, Jialei; Chang, Jianhua; Wu, Xianghua; Hu, Aiqun

    2014-07-01

    Mutations of epidermal growth factor receptor (EGFR) gene are good predictors of response to treatment with EGFR tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer (NSCLC). It is well established that classic mutations, such as in-frame deletions in exon 19 and the point mutation L858R in exon 21, are associated with high sensitivity to EGFR TKIs. Though mutations in exon 20 are almost correlated with EGFR-TKIs resistance, the awareness that they might confer sensitivity to TKI treatment should be emphasized. Herein, we describe a novel mutation in exon 20 of EGFR in a Chinese male non-smoker, who was diagnosed with stage IV lung adenocarcinoma and characterized by the codon 769 point mutation GTG>GCG, which translates into alanine instead of valine (p.V769A). In this case, the patient showed a good clinical response to erlotinib after paclitaxel/cisplatin first-line and docetaxel second-line chemotherapies. Therefore, we suggest that this rare mutation (p.V769A) may be a sensitive EGFR mutation in NSCLC. The identification of novel EGFR mutations provides new predictive biomarkers for TKI treatment and is essential to the successful use of targeted therapies.

  3. EGFR Exon 20 Insertion/Duplication Mutations Characterize Fibrous Hamartoma of Infancy.

    PubMed

    Park, Jason Y; Cohen, Cynthia; Lopez, Dania; Ramos, Erica; Wagenfuehr, Jennifer; Rakheja, Dinesh

    2016-12-01

    Fibrous hamartoma of infancy (FHI) is a benign mesenchymal tumor histologically characterized by a mixture of intersecting fascicles of fibroblasts/myofibroblasts in collagenous stroma, nests of primitive oval or stellate cells in basophilic mucoid stroma, and mature adipose tissue. We hypothesized that FHI, because of histologic overlap with mesenchymal overgrowth tumors seen in CLOVES (Congenital Lipomatous Overgrowth with Vascular, Epidermal, Skeletal anomalies) and Proteus syndromes, may harbor mutations in signaling pathways associated with cellular proliferation. Formalin-fixed paraffin-embedded material from a discovery set of 4 cases of FHI was investigated by targeted next-generation sequencing of a panel of cancer-associated genes. The results were confirmed by targeted Sanger sequencing of EGFR exon 20. A validation set of 8 cases of FHI and 10 cases of other pediatric fatty tumors were investigated by targeted Sanger sequencing of EGFR exon 20. All 12 cases of FHI, and none of the 10 control tumors, showed EGFR exon 20 insertion/duplication mutations. This is the first report of molecular aberrations in FHI. The consistent occurrence of EGFR exon 20 insertion/duplication mutations in 100% of cases of FHI studied suggests that they must play a principal role in the pathogenesis of FHI, likely by conferring a potential for growth and local infiltration. Although surgical treatment will remain the mainstay of FHI treatment, tyrosine kinase inhibitors may have an adjunctive role in cases that are difficult to resect.

  4. Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

    PubMed

    Nagao, Kazuaki; Togawa, Naoyuki; Fujii, Katsunori; Uchikawa, Hideki; Kohno, Yoichi; Yamada, Masao; Miyashita, Toshiyuki

    2005-11-15

    Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

  5. LncRNA expression profiles of EGFR exon 19 deletions in lung adenocarcinoma ascertained by using microarray analysis.

    PubMed

    Wang, Yumin; Chen, Wei; Chen, Jie; Pan, Qinshi; Pan, Jingye

    2014-09-01

    Studies showed that long chain non-coding RNAs (lncRNAs) involved in the development and progression of lung cancer. However, the mechanisms of EGFR exon 19 deletion in lung adenocarcinoma were unclear. Lung adenocarcinoma was divided into EGFR exon 19 deletion group and EGFR wild-type group. We studied the differential expression profiles of lncRNAs in EGFR exon 19 deletion in lung adenocarcinoma by high-throughput microarray. Using abundant and varied probes, we were able to assess 30,586 lncRNAs and 26,109 mRNAs in our microarray. Compared with the wild-type EGFR, we found that 1,533 lncRNAs and 1,406 mRNAs were differentially expressed (≥ twofold change) in EGFR exon 19 deletion in lung adenocarcinoma, indicating that many lncRNAs were significantly upregulated or downregulated in EGFR exon 19 deletion in lung adenocarcinoma. The 10 lncRNAs were aberrantly expressed in EGFR exon 19 deletion in lung adenocarcinoma compared with wild-type EGFR group validated by real-time RT-PCR. Among these, RP11-325I22.2 and LOC440905 were the most aberrantly expressed in 20 cases of EGFR exon 19 deletion in lung adenocarcinoma samples by real-time RT-PCR. Our study showed lncRNAs expression pattern in EGFR exon 19 deletion in lung adenocarcinoma by microarray. RP11-325I22.2 and LOC440905 might play an important role in the mechanism of EGFR exon 19 deletion in lung adenocarcinoma. The study may provide a new mechanism of EGFR exon 19 deletion in lung adenocarcinoma.

  6. 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.

  7. 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

  8. Exonic deletions of mismatch repair genes MLH1 and MSH2 correlate with prognosis and protein expression levels in malignant melanomas.

    PubMed

    Korabiowska, Monika; Brinck, Ulrich; Stachura, Jerzy; Jawien, Jacek; Hasse, Frank Michael; Cordon-Cardos, Carlos; Fischer, Gösta

    2006-01-01

    The mutations of MLH1 and MSH2 have been reported to be responsible for malignant transformation and tumour progression in several sporadic tumours. Eighty-six primary malignant melanomas with known follow-up were investigated. Point mutations of DNA mismatch repair MLH1 and MSH2 in malignant melanomas were not found. Exon 12 (MSH2) was not present in 26 out of the 86 melanomas and exon 13 (MSH2) was lost in 25 of the tumours. The loss of exon 15 (MLH1) was observed in 22 out of the 86 tumours and the loss of exon 16 (MLH1) in 24 melanomas. The loss of exons correlated strongly with the loss of MLH1 and MSH2 protein expression. In multivariate analysis, including all 4 exons and expressions of MLH1 and MSH2, prognostic significance was found only for loss of exon 12 (MSH2) and loss of exon 15 (MLH1).

  9. 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

  10. 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

  11. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    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.

  12. An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs.

    PubMed

    Fyfe, John C; Hemker, Shelby L; Venta, Patrick J; Fitzgerald, Caitlin A; Outerbridge, Catherine A; Myers, Sherry L; Giger, Urs

    2013-08-01

    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 2.9Mb 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 the 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.

  13. 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

  14. 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.

  15. Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

    PubMed

    Yuan, Feng-Jie; Zhu, Dan-Hua; Tan, Yuan-Yuan; Dong, De-Kun; Fu, Xu-Jun; Zhu, Shen-Long; Li, Bai-Quan; Shu, Qing-Yao

    2012-11-01

    Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

  16. TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML.

    PubMed

    Mohamed, Aminetou Mint; Balsat, Marie; Koering, Catherine; Maucort-Boulch, Delphine; Boissel, Nicolas; Payen-Gay, Lea; Cheok, Meyling; Mortada, Hussein; Auboeuf, Didier; Pinatel, Christiane; El-Hamri, Mohamed; Tigaud, Isabelle; Hayette, Sandrine; Dumontet, Charles; Cros, Emeline; Flandrin-Gresta, Pascale; Nibourel, Olivier; Preudhomme, Claude; Thomas, Xavier; Nicolini, Franck-Emmanuel; Solly, Françoise; Guyotat, Denis; Campos, Lydia; Michallet, Mauricette; Ceraulo, Antony; Mortreux, Franck; Wattel, Eric

    2017-01-16

    In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2S(high) patients were found to be younger than TET2(low) patients without a difference in the rate of complete remission. However, TET2E2S(high) patients exhibited a significantly lower CIR (p<10(-4)). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

  17. EGFR Exon-Level Biomarkers of the Response to Bevacizumab/Erlotinib in Non-Small Cell Lung Cancer

    PubMed Central

    Baty, Florent; Rothschild, Sacha; Früh, Martin; Betticher, Daniel; Dröge, Cornelia; Cathomas, Richard; Rauch, Daniel; Gautschi, Oliver; Bubendorf, Lukas; Crowe, Susanne; Zappa, Francesco; Pless, Miklos; Brutsche, Martin

    2013-01-01

    Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood. PMID:24039832

  18. 2′-O-Methyl RNA/Ethylene-Bridged Nucleic Acid Chimera Antisense Oligonucleotides to Induce Dystrophin Exon 45 Skipping

    PubMed Central

    Lee, Tomoko; Awano, Hiroyuki; Yagi, Mariko; Matsumoto, Masaaki; Watanabe, Nobuaki; Goda, Ryoya; Koizumi, Makoto; Takeshima, Yasuhiro; Matsuo, Masafumi

    2017-01-01

    Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease characterized by dystrophin deficiency from mutations in the dystrophin gene. Antisense oligonucleotide (AO)-mediated exon skipping targets restoration of the dystrophin reading frame to allow production of an internally deleted dystrophin protein with functional benefit for DMD patients who have out-of-frame deletions. After accelerated US approval of eteplirsen (Exondys 51), which targets dystrophin exon 51 for skipping, efforts are now focused on targeting other exons. For improved clinical benefits, this strategy requires more studies of the delivery method and modification of nucleic acids. We studied a nucleotide with a 2′-O,4′-C-ethylene-bridged nucleic acid (ENA), which shows high nuclease resistance and high affinity for complementary RNA strands. Here, we describe the process of developing a 2′-O-methyl RNA(2′-OMeRNA)/ENA chimera AO to induce dystrophin exon 45 skipping. One 18-mer 2′-OMeRNA/ENA chimera (AO85) had the most potent activity for inducing exon 45 skipping in cultured myotubes. AO85 was administered to mdx mice without significant side effects. AO85 transfection into cultured myotubes from 13 DMD patients induced exon 45 skipping in all samples at different levels and dystrophin expression in 11 patients. These results suggest the possible efficacy of AO-mediated exon skipping changes in individual patients and highlight the 2′-OMeRNA/ENA chimera AO as a potential fundamental treatment for DMD. PMID:28208626

  19. First report of a pathogenic mutation on exon 24 of the NOTCH3 gene in a CADASIL family.

    PubMed

    Valenti, Raffaella; Bianchi, Silvia; Pescini, Francesca; D'Eramo, Camilla; Inzitari, Domenico; Dotti, Maria Teresa; Pantoni, Leonardo

    2011-09-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetically transmitted small vessel disease clinically characterized by migraine, recurrent subcortical strokes, and cognitive and mood disorders. Pathogenic mutations are located on any of the exons of the NOTCH3 gene coding for epidermal-growth factor (EGF)-like repeats of the extracellular domain of the NOTCH3 receptor. Because the gene is large and the mutations cluster on some exons, many laboratories restrict the analysis to these exons. We report the first missense mutation involving exon 24 and causing CADASIL in a 64-year-old man. The patient was admitted to the hospital for a loss of consciousness accompanied by profuse sweating. On examination, some parkinsonian features were present. Over the last 4 years, he had developed postural instability and gait disturbances with repeated falls, behavioral disorders, and cognitive impairment. A diagnostic hypothesis of atypical parkinsonism had been advanced. The presence of multiple subcortical lacunar infarcts and leukoencephalopathy extended to the external capsule on cerebral MRI suggested the presence of CADASIL. The diagnosis was confirmed by finding a heterozygous mutation leading to a cysteine substitution on exon 24 of the NOTCH3 gene. One proband's brother, who had progressive gait disturbances, unilateral action tremor and bradykinesia, and an asymptomatic niece also resulted affected. This report underlines that when CADASIL is suspected the genetic analysis should be performed on all the NOTCH3 exons coding for EGF-like repeats including exon 24 and confirms that CADASIL may have heterogeneous phenotypes.

  20. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications.

    PubMed

    Yasuda, Hiroyuki; Kobayashi, Susumu; Costa, Daniel B

    2012-01-01

    Lung cancer is the leading cause of cancer-related death. The identification of epidermal growth factor receptor (EGFR) somatic mutations defined a new, molecularly classified subgroup of non-small-cell lung cancer (NSCLC). Classic EGFR activating mutations, such as inframe deletions in exon 19 or the Leu858Arg (L858R) point mutation in exon 21 are associated with sensitivity to first generation quinazoline reversible EGFR tyrosine kinase inhibitors (TKIs). EGFR exon 20 insertion mutations, which are typically located after the C-helix of the tyrosine kinase domain of EGFR, may account for up to 4% of all EGFR mutations. Preclinical models have shown that the most prevalent EGFR exon 20 insertion mutated proteins are resistant to clinically achievable doses of reversible (gefitinib, erlotinib) and irreversible (neratinib, afatinib, PF00299804) EGFR TKIs. Growing clinical experience with patients whose tumours harbour EGFR exon 20 insertions corresponds with the preclinical data; very few patients have had responses to EGFR TKIs. Despite the prevalence and biological importance of EEGFR exon 20 insertions, few reports have summarised all preclinical and clinical data on these mutations. Here, we review the literature and provide an update with an emphasis on the structural, molecular, and clinical implications of EGFR exon 20 insertions.

  1. 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

  2. PIK3CA mutations in the kinase domain (exon 20) of uterine endometrial adenocarcinomas are associated with adverse prognostic parameters.

    PubMed

    Catasus, Lluis; Gallardo, Alberto; Cuatrecasas, Miriam; Prat, Jaime

    2008-02-01

    Mutations of the oncogene PIK3CA occur frequently in endometrial carcinomas, but their prognostic significance is unclear. To determine the clinicopathological and molecular implications of these mutations, PIK3CA status was investigated in 109 endometrial (102 endometrioid and 7 mixed) carcinomas and the results were compared with clinicopathological parameters associated with prognosis. Tumors were also investigated for microsatellite instability and PTEN, beta-catenin gene (CTNNB1), K-RAS, and B-RAF mutations. We found 35 PIK3CA somatic missense mutations in 32 (29%) endometrial carcinomas. Eighteen mutations occurred in exon 20 (kinase domain), and 17 in exon 9 (helical domain). Almost all mutated tumors were pure endometrioid adenocarcinomas. All tumors with PIK3CA mutations exhibited myometrial invasion (P=0.032). Lymphovascular invasion was found more frequently in mutated (28%) than nonmutated carcinomas (18%). Histological grade varied significantly according to the location of the PIK3CA mutations whether in exon 9 or exon 20 (P=0.033). The frequency of exon 9 mutations was higher in grade 1 carcinomas (57%) than in grade 2 (29%) or grade 3 (14%) tumors. Conversely, mutations in exon 20 were more common in grade 3 (60%) than in grade 2 (20%) or grade 1 (20%) carcinomas. None of the tumors confined to the endometrium (stage IA) had PIK3CA mutations. Furthermore, whereas 64% of adenocarcinomas with exon 9 mutations had invaded < or =(1/2) of the myometrial thickness (stage IB), 73% of tumors with exon 20 mutations had either deeper myometrial invasion (stage IC) or cervical involvement (stage II) (P=0.045). PIK3CA mutations coexisted with microsatellite instability and mutations in PTEN, CTNNB1, K-RAS, and B-RAF genes. These results favor that PIK3CA mutations are associated with myometrial invasion and, moreover, that tumors harboring PIK3CA mutations in exon 20 are frequently high-grade, deeply invasive endometrial carcinomas that tend to exhibit

  3. Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping

    PubMed Central

    Paik, Paul K.; Drilon, Alexander; Fan, Pang-Dian; Yu, Helena; Rekhtman, Natasha; Ginsberg, Michelle S.; Borsu, Laetitia; Schultz, Nikolaus; Berger, Michael F.; Rudin, Charles M.; Ladanyi, Marc

    2015-01-01

    Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, deletion of the juxtamembrane domain containing the Cbl E3-ubiquitin ligase binding site, and decreased turnover of the resultant aberrant MET protein, were previously reported to be oncogenic in preclinical models. We now report responses to the MET inhibitors crizotinib and cabozantinib in four patients with stage IV lung adenocarcinomas harboring mutations leading to MET exon 14 skipping, highlighting a new therapeutic strategy for the 4% of lung adenocarcinoma patients whose tumors harbor this previously underappreciated genetic alteration. PMID:25971939

  4. Domain mapping of the retinal cyclic GMP phosphodiesterase gamma-subunit. Function of the domains encoded by the three exons of the gamma-subunit gene.

    PubMed

    Takemoto, D J; Hurt, D; Oppert, B; Cunnick, J

    1992-02-01

    Retinal rod-outer-segment phosphodiesterase (PDE) is a heterotetramer consisting of two similar, but not identical, catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). Previously, we have reported that the site of PDE alpha/beta interaction with PDE gamma is located within residues 54-87 [Cunnick, Hurt, Oppert, Sakamoto & Takemoto (1990) Biochem. J. 271, 721-727]. The site for PDE gamma interaction with transducin alpha (T alpha) was found to encompass residues 24-45 of PDE gamma [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. In order to identify binding sites and other functional domains of PDE gamma, the three peptides which are encoded by the three exons of the PDE gamma gene were synthesized chemically. These exons encode for residues 1-49, 50-62 and 63-87 of bovine PDE gamma [Piriev, Purishko, Khramtsov & Lipkin (1990) Dokl. Akad. Nauk. SSSR 315, 229-230]. The peptide encompassing residues 63-87 was inhibitory in a PDE assay, whereas peptides 1-49 and 50-62 had no effect. However, both peptides 1-49 and 63-87 bound to PDE alpha/beta in a solid-phase binding assay. Only peptide 1-49 bound to T alpha.GTP[S] (GTP[S] is guanosine 5'-[gamma-thio]triphosphate). These data confirm that the inhibitory region of PDE gamma is encoded by exon 3 (residues 63-87), whereas a separate binding site for PDE alpha/beta and for T alpha.GTP[S] is encoded by exon 1 (residues 1-49). To study further the structure-function relationship of PDE gamma, this entire protein and two mutants were chemically synthesized. One mutant (-CT) lacked residues 78-87, whereas another replaced tyrosine-84 with glycine (TYR-84). Whereas the synthetic PDE gamma inhibited PDE alpha/beta catalytic activity, the -CT and TVR-84 mutants did not. All three synthetic proteins bound to both PDE alpha/beta and and T alpha.GTP[S]. These data confirm the presence of an alternative binding site on PDE gamma and demonstrate the importance of tyrosine

  5. 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

  6. Exonic deletions of AUTS2 in Chinese patients with developmental delay and intellectual disability.

    PubMed

    Fan, Yanjie; Qiu, Wenjuan; Wang, Lili; Gu, Xuefan; Yu, Yongguo

    2016-02-01

    Genomic rearrangements involving dosage change of genes have been implicated in a range of developmental disorders. Increasing evidences suggest copy number variations (CNVs) of autism susceptibility candidate gene 2 (AUTS2) are associated with a syndromic form of developmental delay and intellectual disability. However, the genetic and clinical profiles involving AUTS2 variations have not been fully characterized in Asian patients yet, and the outcome of treatments has not been reported. Here we report de novo exonic deletions of AUTS2 detected by chromosomal microarray analysis (CMA) in three Chinese children referred to the clinic for developmental delay, including two deletions involving only exon 6 (98.4 and 262 kb, respectively) and one deletion involving the C-terminal of AUTS2 (2147 kb). The phenotypic presentations of these three patients were described and compared with previous cases in literature. In addition, we presented the outcome of hormonal treatment for short stature in one patient.

  7. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice

    PubMed Central

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J. A.; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose–fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  8. 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.

  9. EXONIC SINE INSERTION IN STK38L CAUSES CANINE EARLY RETINAL DEGENERATION (erd)

    PubMed Central

    Goldstein, Orly; Kukekova, Anna V.; Aguirre, Gustavo D.; Acland, Gregory M.

    2010-01-01

    Fine mapping followed by candidate gene analysis of erd - a canine hereditary retinal degeneration characterized by aberrant photoreceptor development - established that the disease cosegregates with a SINE insertion in exon 4 of the canine STK38L/NDR2 gene. The mutation removes exon 4 from STK38L transcripts and is predicted to remove much of the N-terminus from the translated protein, including binding sites for S100B and Mob Proteins, part of the protein kinase domain, and a Thr-75 residue critical for autophosphorylation. Although known to have roles in neuronal cell function, the STK38L pathway has not previously been implicated in normal or abnormal photoreceptor development. Loss of STK38L function in erd provides novel potential insights into the role of the STK38L pathway in neuronal and photoreceptor cell function, and suggests that genes in this pathway need to be considered as candidate genes for hereditary retinal degenerations. PMID:20887780

  10. Exonic SINE insertion in STK38L causes canine early retinal degeneration (erd).

    PubMed

    Goldstein, Orly; Kukekova, Anna V; Aguirre, Gustavo D; Acland, Gregory M

    2010-12-01

    Fine mapping followed by candidate gene analysis of erd - a canine hereditary retinal degeneration characterized by aberrant photoreceptor development - established that the disease cosegregates with a SINE insertion in exon 4 of the canine STK38L/NDR2 gene. The mutation removes exon 4 from STK38L transcripts and is predicted to remove much of the N terminus from the translated protein, including binding sites for S100B and Mob proteins, part of the protein kinase domain, and a Thr-75 residue critical for autophosphorylation. Although known to have roles in neuronal cell function, the STK38L pathway has not previously been implicated in normal or abnormal photoreceptor development. Loss of STK38L function in erd provides novel potential insights into the role of the STK38L pathway in neuronal and photoreceptor cell function, and suggests that genes in this pathway need to be considered as candidate genes for hereditary retinal degenerations.

  11. 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

  12. Gene duplication followed by exon structure divergence substitutes for alternative splicing in zebrafish.

    PubMed

    Lambert, Matthew J; Olsen, Kyle G; Cooper, Cynthia D

    2014-08-10

    In this study we report novel findings regarding the evolutionary relationship between gene duplication and alternative splicing, two processes that increase proteomic diversity. By studying teleost fish, we find that gene duplication followed by exon structure divergence between paralogs, but not gene duplication alone, leads to a significant reduction in alternative splicing, as measured by both the proportion of genes that undergo alternative splicing as well as mean number of transcripts per gene. Additionally, we show that this effect is independent of gene family size and gene function. Furthermore, we provide evidence that the reduction in alternative splicing may be due to the partitioning of ancestral splice forms among the duplicate genes - a form of subfunctionalization. Taken together these results indicate that exon structure evolution subsequent to gene duplication may be a common substitute for alternative splicing.

  13. 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.

  14. 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

  15. Single-Nucleotide Polymorphism on Exon 17 of Insulin Receptor Gene Influences Insulin Resistance in PCOS: A Pilot Study on North Indian Women.

    PubMed

    Gangopadhyay, Sukanya; Agrawal, Nitin; Batra, Aruna; Kabi, Bhaskar Charan; Gupta, Akash

    2016-04-01

    Polycystic ovarian syndrome (PCOS), a major cause of infertility, is also strongly associated with insulin resistance. Defects in insulin receptor signaling are considered as one of the major molecular pathogeneses for insulin resistance. To investigate the possible mechanism of this signaling defect at genetic level, single-nucleotide polymorphism (SNP) [His 1085 C/T] at the exon 17 of insulin receptor gene (INSR) was studied in this pilot study. Polymerase chain reaction was performed on leucocytic DNA of women diagnosed with PCOS, selected from the outpatient department of Safdarjung Hospital, New Delhi, using suitable primer to amplify a region on INSR. An equal number of age-matched healthy women were selected as controls. SNP analysis was performed with restriction enzyme length polymorphism technique using Pm II enzyme. Serum insulin level was measured by ELISA kit and HOMA-IR was calculated mathematically. A higher frequency of the CC genotype was observed in PCOS women than in controls. Also, HOMA-IR, a tool for estimating insulin resistance, was significantly high in PCOS women with the CC genotype. C1008T SNP at exon 17 of INSR is associated with insulin resistance in Indian women with PCOS. Presence of CC genotype (C1085T) could be developed as a marker for insulin resistance and metabolic complications in PCOS women.

  16. Hypoxia inducible factor-1 mediates the expression of the immune checkpoint HLA-G in glioma cells through hypoxia response element located in exon 2.

    PubMed

    Yaghi, Layale; Poras, Isabelle; Simoes, Renata T; Donadi, Eduardo A; Tost, Jörg; Daunay, Antoine; de Almeida, Bibiana Sgorla; Carosella, Edgardo D; Moreau, Philippe

    2016-09-27

    HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G. We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2'deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G. Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at -966 bp in the 5'UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus.

  17. Hypoxia inducible factor-1 mediates the expression of the immune checkpoint HLA-G in glioma cells through hypoxia response element located in exon 2

    PubMed Central

    Yaghi, Layale; Poras, Isabelle; Simoes, Renata T.; Donadi, Eduardo A.; Tost, Jörg; Daunay, Antoine; de Almeida, Bibiana Sgorla; Carosella, Edgardo D.; Moreau, Philippe

    2016-01-01

    HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G. We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2′deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G. Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at −966 bp in the 5′UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus. PMID:27577073

  18. 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.

  19. Direct Competition between hnRNP C and U2AF65 Protects the Transcriptome from the Exonization of Alu Elements

    PubMed Central

    Zarnack, Kathi; König, Julian; Tajnik, Mojca; Martincorena, Iñigo; Eustermann, Sebastian; Stévant, Isabelle; Reyes, Alejandro; Anders, Simon; Luscombe, Nicholas M.; Ule, Jernej

    2013-01-01

    Summary There are ∼650,000 Alu elements in transcribed regions of the human genome. These elements contain cryptic splice sites, so they are in constant danger of aberrant incorporation into mature transcripts. Despite posing a major threat to transcriptome integrity, little is known about the molecular mechanisms preventing their inclusion. Here, we present a mechanism for protecting the human transcriptome from the aberrant exonization of transposable elements. Quantitative iCLIP data show that the RNA-binding protein hnRNP C competes with the splicing factor U2AF65 at many genuine and cryptic splice sites. Loss of hnRNP C leads to formation of previously suppressed Alu exons, which severely disrupt transcript function. Minigene experiments explain disease-associated mutations in Alu elements that hamper hnRNP C binding. Thus, by preventing U2AF65 binding to Alu elements, hnRNP C plays a critical role as a genome-wide sentinel protecting the transcriptome. The findings have important implications for human evolution and disease. PMID:23374342

  20. 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

  1. Transcriptional enhancers in protein-coding exons of vertebrate developmental genes.

    PubMed

    Ritter, Deborah I; Dong, Zhiqiang; Guo, Su; Chuang, Jeffrey H

    2012-01-01

    Many conserved noncoding sequences function as transcriptional enhancers that regulate gene expression. Here, we report that protein-coding DNA also frequently contains enhancers functioning at the transcriptional level. We tested the enhancer activity of 31 protein-coding exons, which we chose based on strong sequence conservation between zebrafish and human, and occurrence in developmental genes, using a Tol2 transposable GFP reporter assay in zebrafish. For each exon we measured GFP expression in hundreds of embryos in 10 anatomies via a novel system that implements the voice-recognition capabilities of a cellular phone. We find that 24/31 (77%) exons drive GFP expression compared to a minimal promoter control, and 14/24 are anatomy-specific (expression in four anatomies or less). GFP expression driven by these coding enhancers frequently overlaps the anatomies where the host gene is expressed (60%), suggesting self-regulation. Highly conserved coding sequences and highly conserved noncoding sequences do not significantly differ in enhancer activity (coding: 24/31 vs. noncoding: 105/147) or tissue-specificity (coding: 14/24 vs. noncoding: 50/105). Furthermore, coding and noncoding enhancers display similar levels of the enhancer-related histone modification H3K4me1 (coding: 9/24 vs noncoding: 34/81). Meanwhile, coding enhancers are over three times as likely to contain an H3K4me1 mark as other exons of the host gene. Our work suggests that developmental transcriptional enhancers do not discriminate between coding and noncoding DNA and reveals widespread dual functions in protein-coding DNA.

  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. Suppression of 5' splice-sites through multiple exonic motifs by hnRNP L.

    PubMed

    Loh, Tiing Jen; Choi, Namjeong; Moon, Heegyum; Jang, Ha Na; Liu, Yongchao; Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2017-03-01

    Selection of 5' splice-sites (5'SS) in alternative splicing plays an important role in gene regulation. Although regulatory mechanisms of heterogeneous nuclear ribonucleoprotein L (hnRNP L), a well-known splicing regulatory protein, have been studied in a substantial level, its role in 5'SS selection is not thoroughly defined. By using a KLF6 pre-mRNA alternative splicing model, we demonstrate in this report that hnRNP L inhibits proximal 5'SS but promotes two consecutive distal 5'SS splicing, antagonizing SRSF1 roles in KLF6 pre-mRNA splicing. In addition, three consecutive CA-rich sequences in a CA cassette immediately upstream of the proximal 5'SS are all required for hnRNP L functions. Importantly, the CA-cassette locations on the proximal exon do not affect hnRNP L roles. We further show that the proximal 5'SS but not the two distal 5'SSs are essential for hnRNP L activities. Notably, in a Bcl-x pre-mRNA model that contains two alternative 5'SS but includes CA-rich elements at distal exon, we demonstrate that hnRNP L also suppresses nearby 5'SS activation. Taken together, we conclude that hnRNP L suppresses 5'SS selection through multiple exonic motifs.

  4. 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

  5. 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

  6. Antisense-mediated Exon Skipping Decreases Tau Protein Expression: A Potential Therapy For Tauopathies.

    PubMed

    Sud, Reeteka; Geller, Evan T; Schellenberg, Gerard D

    2014-07-29

    In Alzheimer's disease, progressive supranuclear palsy, and a number of other neurodegenerative diseases, the microtubule associated protein tau aggregates to form intracellular neurofibrillary tangles and glial tangles, abnormal structures that are part of disease pathogenesis. Disorders with aggregated tau are called tauopathies. Presently, there are no disease-modifying treatments for this disease class. Tau is encoded by the MAPT gene. We propose that reducing MAPT expression and thus the amount of tau protein made could prevent aggregation, and potentially be an approach to treat tauopathies. We tested 31 morpholinos, complementary to the sense strand of the MAPT gene to identify oligonucleotides that can downregulate MAPT expression and reduce the amount of tau protein produced. Oligonucleotides were tested in human neuroblastoma cell lines SH-SY5Y and IMR32. We identified several morpholinos that reduced MAPT mRNA expression up to 50% and tau protein levels up to ~80%. The two most potent oligonucleotides spanned the 3' boundary of exons 1 and 5, masking the 5'-splice sites of these exons. Both morpholinos induced skipping of the targeted exons. These in vitro findings were confirmed in mice transgenic for the entire human MAPT gene and that express human tau protein. These studies demonstrate the feasibility of using modified oligonucleotides to alter tau expression.

  7. 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

  8. 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

  9. RBM5/Luca-15/H37 regulates Fas alternative splice site pairing after exon definition.

    PubMed

    Bonnal, Sophie; Martínez, Concepción; Förch, Patrik; Bachi, Angela; Wilm, Matthias; Valcárcel, Juan

    2008-10-10

    RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3' splice site recognition and regulates alternative splicing of apoptosis-related genes, including the Fas receptor, switching between isoforms with antagonistic functions in programmed cell death. In contrast with classical mechanisms of splicing regulation, RBM5 does not affect early events of splice site recognition that lead to Fas exon 6 definition. Instead, RBM5 inhibits the transition between prespliceosomal complexes assembled around exon 6 to mature spliceosomes assembled on the flanking introns and promotes sequence-specific pairing of the distal splice sites. An OCRE domain important for RBM5 function contacts components of the U4/5/6 tri-snRNP, consistent with the idea that RBM5 modulates splice site pairing after prespliceosome assembly and exon definition.

  10. Exon- and contraction-dependent functions of titin in sarcomere assembly.

    PubMed

    Shih, Yu-Huan; Dvornikov, Alexey V; Zhu, Ping; Ma, Xiao; Kim, Maengjo; Ding, Yonghe; Xu, Xiaolei

    2016-12-15

    Titin-truncating variants (TTNtvs) are the major cause of dilated cardiomyopathy (DCM); however, allelic heterogeneity (TTNtvs in different exons) results in variable phenotypes, and remains a major hurdle for disease diagnosis and therapy. Here, we generated a panel of ttn mutants in zebrafish. Four single deletion mutants in ttn.2 or ttn.1 resulted in four phenotypes and three double ttn.2/ttn.1 mutants exhibited more severe phenotypes in somites. Protein analysis identified ttn(xu071) as a near-null mutant and the other six mutants as hypomorphic alleles. Studies of ttn(xu071) uncovered a function of titin in guiding the assembly of nascent myofibrils from premyofibrils. By contrast, sarcomeres were assembled in the hypomorphic ttn mutants but either became susceptible to biomechanical stresses such as contraction or degenerated during development. Further genetic studies indicated that the exon usage hypothesis, but not the toxic peptide or the Cronos hypothesis, could account for these exon-dependent effects. In conclusion, we modeled TTNtv allelic heterogeneity during development and paved the way for future studies to decipher allelic heterogeneity in adult DCM.

  11. MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy

    PubMed Central

    Pilotto, Sara; Gkountakos, Anastasios; Carbognin, Luisa; Scarpa, Aldo; Tortora, Giampaolo

    2017-01-01

    The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients’ subgroup, are explored. PMID:28164087

  12. 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.

  13. Identification of a new insertion in exon 20 of EGFR in a woman with NSCLC.

    PubMed

    Zupa, Angela; Vita, Giulia; Landriscina, Matteo; Possidente, Luciana; Aieta, Michele; Tartarone, Alfredo; Improta, Giuseppina

    2012-12-01

    Mutations of epidermal growth factor receptor 1 (EGFR) gene occur in about 15 % of all NSCLCs in Western Europe and are frequently located in exons 19 and 21, being associated with high sensitivity to EGFR tyrosine kinase inhibitors (TKIs). By contrast, exon 20 insertions account for up to 10 % of all EGFR mutations and are correlated to EGFR TKI resistance. Herein, we describe a novel mutation in EGFR exon 20 in a female non-smoker bearing a lung adenocarcinoma, characterized by the insertion of a nucleotide triplet GTT, which translates into a protein with an additional Valine between Proline 772 and Histidine 773 (p.P772_H773insV-c.2316_2317insGTT). The patient was treated with cisplatin/pemetrexed 1st-line and docetaxel 2nd-line chemotherapies, reporting a prolonged disease stabilization of 25 months. The identification and the biological and clinical characterization of novel EGFR mutations represent a prerequisite for their wide use as predictive biomarkers for personalized therapy in NSCLC.

  14. SVA retrotransposition in exon 6 of the coagulation factor IX gene causing severe hemophilia B.

    PubMed

    Nakamura, Yuki; Murata, Moe; Takagi, Yuki; Kozuka, Toshihiro; Nakata, Yukiko; Hasebe, Ryo; Takagi, Akira; Kitazawa, Jun-ichi; Shima, Midori; Kojima, Tetsuhito

    2015-07-01

    Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities of the coagulation factor IX gene (F9). Insertion mutations in F9 ranging from a few to more than 100 base pairs account for only a few percent of all hemophilia B cases. We investigated F9 to elucidate genetic abnormalities causing severe hemophilia B in a Japanese subject. We performed PCR-mediated analysis of F9 and identified a large insertion in exon 6. Next, we carried out direct sequencing of a PCR clone of the whole insert using nested deletion by exonuclease III and S1 nuclease. We identified an approximately 2.5-kb SINE-VNTR-Alu (SVA)-F element flanked by 15-bp duplications in the antisense orientation in exon 6. Additionally, we carried out exontrap analysis to assess the effect of this retrotransposition on mRNA splicing. We observed that regular splicing at exons 5 and 6 of F9 was disturbed by the SVA retrotransposition, suggesting that abnormal FIX mRNA may be reduced by nonsense-mediated mRNA decay. In conclusion, this is the first report of SVA retrotransposition causing severe hemophilia B; only five cases of LINE-1 or Alu retrotranspositions in F9 have been reported previously.

  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. MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy.

    PubMed

    Pilotto, Sara; Gkountakos, Anastasios; Carbognin, Luisa; Scarpa, Aldo; Tortora, Giampaolo; Bria, Emilio

    2017-01-01

    The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients' subgroup, are explored.

  17. 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

  18. 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.

  19. ANTIDEPRESSANTS REVERSE CORTICOSTERONE-MEDIATED DECREASE IN BDNF EXPRESSION: DIFFERENTIAL REGULATION OF SPECIFIC EXONS BY ANTIDEPRESSANTS AND CORTICOSTERONE

    PubMed Central

    Dwivedi, Yogesh; Rizavi, Hooriyah S.; Pandey, Ghanshyam N.

    2006-01-01

    Earlier studies have implicated BDNF in stress and in the mechanism of action of antidepressants. It has been shown that antidepressants upregulate, whereas corticosterone downregulates, BDNF expression in rat brain. Whether various classes of antidepressants reverse corticosterone-mediated downregulation of BDNF is unclear. Also not known is how antidepressants or corticosterone regulate BDNF expression. To clarify this, we examined the effects of various classes of antidepressants and corticosterone, alone and in combination, on the mRNA expression of total BDNF and of individual BDNF exons, in rat brain. Normal or corticosterone pellet-implanted (100 mg, 21 days) rats were injected with different classes of antidepressants, fluoxetine, desipramine, or phenelzine, intraperitoneally for 21 days and sacrificed 2 h after the last injection. mRNA expression of total BDNF and of exons I-IV was measured in frontal cortex and hippocampus. Given to normal rats, fluoxetine increased total BDNF mRNA only in hippocampus, whereas desipramine or phenelzine increased BDNF mRNA in both frontal cortex and hippocampus. When specifc exons were examined, desipramine increased expression of exons I and III in both brain areas, whereas phenelzine increased exon I in both frontal cortex and hippocampus but exon IV only in hippocampus. On the other hand, fluoxetine increased only exon II in hippocampus. Corticosterone treatment of normal rats decreased expression of total BDNF mRNA in both brain areas, specifically decreasing exons II and IV. Treatment with desipramine or phenelzine of corticosterone pellet-implanted rats reversed the corticosterone-induced decrease in total BDNF expression in both brain areas; however, fluoxetine reversed the decrease only partially in hippocampus. Interestingly, antidepressant treatment of corticosterone pellet-implanted rats increased only those specific exons that are increased during treatment of normal rats with each particular antidepressant. We

  20. 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.

  1. Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1

    SciTech Connect

    Reisman, D.; Greenberg, M.; Rotter, V. )

    1988-07-01

    To gain insight into how transcription of the human p53 oncogene is controlled, the authors characterized the regulatory regions of the gene. A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5{prime} 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 pSVO-CAT-derived constructs that were transiently introduced into the monkey cell line COS. By this approach they 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. They 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.

  2. Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits

    PubMed Central

    Speed, Haley E.; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M.; Ochoa, Christine F.; Gupta, Natasha; Liu, Shunan

    2015-01-01

    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 (Shank3G). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3G/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 Shank3G/G mouse that was engineered with such future experiments in mind. PMID:26134648

  3. 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

  4. TGF-beta(1) regulation of human AT(1) receptor mRNA splice variants harboring exon 2.

    PubMed

    Martin, Mickey M; Buckenberger, Jessica A; Knoell, Daren L; Strauch, Arthur R; Elton, Terry S

    2006-04-25

    At least four alternatively spliced mRNAs can be synthesized from the human AT(1)R (hAT(1)R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT(1)R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT(1)R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT(1)R mRNA transcripts dramatically reduces hAT(1)R protein levels (nine-fold) and significantly attenuates Ang II responsiveness ( approximately four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT(1)R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT(1)R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF-beta(1) treatment (4ng/ml for 4h) of fibroblasts up-regulated hAT(1)R mRNA splice variants, which harbored exon 2, six-fold. Since AT(1)R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT(1)R protein and possibly limit the potential pathological effects of AT(1)R activation.

  5. In Silico Screening Based on Predictive Algorithms as a Design Tool for Exon Skipping Oligonucleotides in Duchenne Muscular Dystrophy

    PubMed Central

    Echigoya, Yusuke; Mouly, Vincent; Garcia, Luis; Yokota, Toshifumi; Duddy, William

    2015-01-01

    The use of antisense ‘splice-switching’ oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2’O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R2 0.89) and 53 (R2 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each

  6. A Prospective Study in the Rational Design of Efficient Antisense Oligonucleotides for Exon Skipping in the DMD Gene

    PubMed Central

    Wee, Keng Boon; Wang, Jian Li; Chen, Yi Jun; Xiong, Qian Bin; Lai, Poh San; Yee, Woon Chee

    2012-01-01

    Abstract Antisense oligonucleotide (AON)-mediated exon skipping to restore dystrophin expression in Duchenne muscular dystrophy (DMD) therapy shown promise in a number of human clinical trials. Current AON design methods are semi-empirical, involving either trial-and-error and/or preliminary experimentations. Therefore, a rational approach to design efficient AONs to address the wide spectrum of patients' mutations is desirable. Retrospective studies have extracted many AON design variables, but they were not tested prospectively to design AONs for skipping DMD exons. Not only did the variables differ among the various studies, no numerical cutoff for each variable was inferred, which makes their use in AON design difficult. The challenge is to thus select a minimal set of key independent variables that can consistently design efficient AONs. In this prospective study, a novel set of design variables with respective cutoff values was used to design 23 novel AONs, each to skip one of nine DMD exons. Nineteen AONs were found to be efficacious in inducing specific exon skipping (83% of total), of which 14 were considered efficient (61% of total), i.e., they induced exon skipping in >25% of total transcripts. Notably, the satisfactory success rates were achieved by using only three design variables; namely, co-transcriptional binding accessibility of target site, presence of exonic splicing enhancers, and target length. Retrospective analyses revealed that the most efficient AON in every exon targeted has the lowest average cumulative position (ACP) score. Taking the prospective and retrospective studies together, we propose that design guidelines recommend using the ACP score to select the most efficient AON for each exon. PMID:22486275

  7. In silico screening based on predictive algorithms as a design tool for exon skipping oligonucleotides in Duchenne muscular dystrophy.

    PubMed

    Echigoya, Yusuke; Mouly, Vincent; Garcia, Luis; Yokota, Toshifumi; Duddy, William

    2015-01-01

    The use of antisense 'splice-switching' oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2'O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R² 0.89) and 53 (R² 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of

  8. Post-entrapment genome engineering: first exon size does not affect the expression of fusion transcripts generated by gene entrapment.

    PubMed

    Osipovich, Anna B; Singh, Aparna; Ruley, H Earl

    2005-03-01

    Gene trap mutagenesis in mouse embryonic stem cells has been widely used for genome-wide studies of mammalian gene function. However, while large numbers of genes can be disrupted, individual mutations may suffer from limitations due to the structure and/or placement of targeting vector. To extend the utility of gene trap mutagenesis, replaceable 3' [or poly(A)] gene trap vectors were developed that permit sequences inserted in individual entrapment clones to be engineered by Cre-mediated recombination. 3' traps incorporating different drug resistance genes could be readily exchanged, simply by selecting for the drug-resistance gene of the replacement vector. By substituting different 3' traps, we show that otherwise identical fusion genes containing a large first exon (804 nt) are not expressed at appreciably lower levels than genes expressing small first exons (384 and 151 nt). Thus, size appears to have less effect on the expression and processing of first exons than has been reported for internal exons. Finally, a retroviral poly(A) trap (consisting of a RNA polymerase II promoter, a neomycin-resistance gene, and 5'-splice site) typically produced mutagenized clones in which vector sequences spliced to the 3'-terminal exons of cellular transcription units, suggesting strong selection for fusion transcripts that evade nonsense-mediated decay. The efficient exchange of poly(A) traps should greatly extend the utility of mutant libraries generated by gene entrapment and provides new strategies to study the rules that govern the expression of exons inserted throughout the genome.

  9. 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.

  10. Sensitivity of epidermal growth factor receptor and ErbB2 exon 20 insertion mutants to Hsp90 inhibition.

    PubMed

    Xu, W; Soga, S; Beebe, K; Lee, M-J; Kim, Y S; Trepel, J; Neckers, L

    2007-09-17

    The mature epidermal growth factor receptor (EGFR) neither associates with nor requires the molecular chaperone heat-shock protein 90 (Hsp90). Mutations in EGFR exons 18, 19, and 21 confer Hsp90 chaperone dependence. In non-small cell lung cancer (NSCLC), these mutations are associated with enhanced sensitivity to EGFR inhibitors in vitro and with clinical response in vivo. Although less prevalent, insertions in EGFR exon 20 have also been described in NSCLC. These mutations, however, confer resistance to EGFR inhibitors. In NSCLC, exon 20 insertions have also been identified in the EGFR family member ErbB2. Here, we examined the sensitivity of exon 20 insertion mutants to an Hsp90 inhibitor currently in the clinic. Our data demonstrate that both EGFR and ErbB2 exon 20 insertion mutants retain dependence on Hsp90 for stability and downstream-signalling capability, and remain highly sensitive to Hsp90 inhibition. Use of Hsp90 inhibitors should be considered in NSCLC harbouring exon 20 insertions in either EGFR or ErbB2.

  11. Mutation in intron 6 of the hamster Mitf gene leads to skipping of the subsequent exon and creates a novel animal model for the human Waardenburg syndrome type II.

    PubMed Central

    Graw, Jochen; Pretsch, Walter; Löster, Jana

    2003-01-01

    In the course of analysis of ENU-induced mutations in Syrian hamsters, a novel dominant anophthalmic white mutant (Wh(V203)) with hearing loss was recovered. Because of this phenotype and a close linkage to the Tpi gene, the Mitf gene was considered as a candidate gene. In the Mitf cDNA, a deletion of 76 bp covering the entire exon 7 was detected. Further molecular analysis revealed a T --> A exchange 16 bp upstream of the end of intron 6, leading to skipping of exon 7. These 16 bp at the end of intron 6 are identical in hamster, rat, mouse, and humans, indicating high conservation during evolution and a functional importance in splicing. Since the loss of exon 7 changes the open reading frame of the MITF transcript, translation will be stopped after 10 new amino acids. The truncated protein is predicted to contain only a part of the basic region and will miss the two helical domains and the leucine zipper. The Wh(V203) mutation in the Syrian hamster affects the same functional domains of the Mitf transcription factor as the human R124X mutation, causing human Waardenburg syndrome type II. Therefore, the Wh(V203) hamster mutant provides a novel model for this particular syndrome. PMID:12871913

  12. Splicing of two internal and four carboxyl-terminal alternative exons in nonmuscle tropomyosin 5 pre-mRNA is independently regulated during development.

    PubMed

    Dufour, C; Weinberger, R P; Schevzov, G; Jeffrey, P L; Gunning, P

    1998-07-17

    Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251-256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a + 9b, 9a + 9c, 9a + 9d, 9c, and 9d), and the exon combinations 9a + 9c and 9a + 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.

  13. Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation.

    PubMed

    Green, P L; Ross, T M; Chen, I S; Pettiford, S

    1995-01-01

    Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.

  14. A novel exon in the human Ca2+-activated Cl- channel Ano1 imparts greater sensitivity to intracellular Ca2.

    PubMed

    Strege, Peter R; Bernard, Cheryl E; Mazzone, Amelia; Linden, David R; Beyder, Arthur; Gibbons, Simon J; Farrugia, Gianrico

    2015-11-01

    Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electro