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Sample records for 3prime untranslated region

  1. The myotonic dystrophy kinase 3{prime}-untranslated region and its effect on gene expression

    SciTech Connect

    Ang, C.W.Y.; Sabourin, L.A.; Narang, M.A.

    1994-09-01

    Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease involving the expansion of an unstable CTG repeat in the 3{prime}-untranslated (3{prime}-UTR) region of the DM kinase (DMK) gene. Increased levels of mRNA in congenital compared to normal tissue have been shown, suggesting elevated DMK levels may be responsible for the disease phenotype. To study the effect of the DMK 3{prime}UTR on gene expression, a reporter gene system was constructed using the constitutive CMV promoter with the chloramphenicol acetyl transferase (CAT) open reading frame and the DMK 3{prime}UTR containing from 5 repeats up to 90 repeats. Transient transfection into a rhabdomyosarcoma cell line shows a three-fold increase in CAT activity from constructs containing a wildtype 3{prime}UTR (5 and 20 repeats) compared to a control construct containing only a poly(A) signal. Reporter constructs with repeats in the protomutation (50 repeats) and mutation (90 repeats) range show a greater than 10-fold increase over control CAT activity. These results suggest the presence of elements in the DMK 3{prime}UTR capable of conferring increased gene expression. We are currently investigating cell-specific activity of the constructs and conducting deletion mapping to identify regulatory elements in the 3{prime}-UTR.

  2. An AU-rich element in the 3{prime} untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation

    SciTech Connect

    Chen, Qiuyun; Adams, C.C.; Usack, L.

    1995-04-01

    In chloroplasts, the 3{prime} untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3{prime}-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3{prime} IR-containing RNA (3{prime} IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b{sub 6}/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T{sub 1} digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3{prime} IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3{prime} untranslated region, only a single copy, which we have termed box II, appears to be essential for in vivo protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3{prime}-end processing and/or influence the stability of petD mRNA in chloroplasts. 48 refs., 9 figs., 2 tabs.

  3. Untranslated regions (UTRs) orchestrate translation reprogramming in cellular stress responses.

    PubMed

    Sajjanar, Basavaraj; Deb, Rajib; Raina, Susheel Kumar; Pawar, Sachin; Brahmane, Manoj P; Nirmale, Avinash V; Kurade, Nitin P; Manjunathareddy, Gundallahalli B; Bal, Santanu Kumar; Singh, Narendra Pratap

    2017-04-01

    Stress is the result of an organism's interaction with environmental challenges. Regulations of gene expression including translation modulations are critical for adaptation and survival under stress. Untranslated regions (UTRs) of the transcripts play significant roles in translation regulation and continue to raise many intriguing questions in our understanding of cellular stress physiology. IRES (Internal ribosome entry site) and uORF (upstream open reading frame) mediated alternative translation initiations are emerging as unique mechanisms. Recent studies have revealed novel means of mRNAs stabilization in stress granules and their reversible modifications. Differential regulation of select transcripts is possible by the interplay between the adenine/uridine-rich elements (AREs) in 3'UTR with their binding proteins (AUBP) and by microRNA-mediated effects. Coordination of these various mechanisms control translation and thereby enables appropriate responses to environmental stress. In this review, we focus on the role of sequence signatures both at 5' and 3'UTRs in translation reprogramming during cellular stress responses.

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

    SciTech Connect

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

    1991-04-01

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

  5. UTRdb: a specialized database of 5'- and 3'-untranslated regions of eukaryotic mRNAs.

    PubMed Central

    Pesole, G; Liuni, S; Grillo, G; Saccone, C

    1998-01-01

    The important role the untranslated regions of eukaryotic mRNAs may play in gene regulation and expression is now widely acknowledged. For this reason we developed UTRdb, a specialized database of 5'- and 3'-untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases, including the presence of functional patterns already demonstrated by experimental analysis to have some functional role. A collection of such patterns is being collected in UTRsite database (http://bio-www.ba.cnr.it:8000/srs5/) which can also be used with appropriate computational tools to detect known functional patterns contained in mRNA untranslated regions. PMID:9399833

  6. Dom34 Rescues Ribosomes in 3´ Untranslated Regions

    PubMed Central

    Guydosh, Nicholas R.; Green, Rachel

    2014-01-01

    SUMMARY Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3´ UTRs. These ribosomes appear to gain access to the 3 UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them. PMID:24581494

  7. Dom34 rescues ribosomes in 3' untranslated regions.

    PubMed

    Guydosh, Nicholas R; Green, Rachel

    2014-02-27

    Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here, we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3' UTRs. These ribosomes appear to gain access to the 3' UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them.

  8. Alternative 5' untranslated regions are involved in expression regulation of human heme oxygenase-1.

    PubMed

    Kramer, Marcel; Sponholz, Christoph; Slaba, Monique; Wissuwa, Bianka; Claus, Ralf A; Menzel, Uwe; Huse, Klaus; Platzer, Matthias; Bauer, Michael

    2013-01-01

    The single nucleotide polymorphism rs2071746 and a (GT)n microsatellite within the human gene encoding heme oxygenase-1 (HMOX1) are associated with incidence or outcome in a variety of diseases. Most of these associations involve either release of heme or oxidative stress. Both polymorphisms are localized in the promoter region, but previously reported correlations with heme oxygenase-1 expression remain not coherent. This ambiguity suggests a more complex organization of the 5' gene region which we sought to investigate more fully. We evaluated the 5' end of HMOX1 and found a novel first exon 1a placing the two previously reported polymorphisms in intronic or exonic positions within the 5' untranslated region respectively. Expression of exon 1a can be induced in HepG2 hepatoma cells by hemin and is a repressor of heme oxygenase-1 translation as shown by luciferase reporter assays. Moreover, minigene approaches revealed that the quantitative outcome of alternative splicing within the 5' untranslated region is affected by the (GT)n microsatellite. This data supporting an extended HMOX1 gene model and provide further insights into expression regulation of heme oxygenase-1. Alternative splicing within the HMOX1 5' untranslated region contributes to translational regulation and is a mechanistic feature involved in the interplay between genetic variations, heme oxygenase-1 expression and disease outcome.

  9. UTRdb: a specialized database of 5' and 3' untranslated regions of eukaryotic mRNAs.

    PubMed Central

    Pesole, G; Liuni, S; Grillo, G; Ippedico, M; Larizza, A; Makalowski, W; Saccone, C

    1999-01-01

    The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb (http://bigarea.area.ba.cnr.it:8000/BioWWW/#U TRdb), a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. PMID:9847176

  10. HLA-G coding region and 3'untranslated region (3'UTR) in two Chinese Han populations.

    PubMed

    Wang, Wen Yi; Tian, Wei; Liu, Xue Xiang; Li, Li Xin

    2016-08-01

    In this study, exons 2-4 and 3'untranslated region (3'UTR) of human leukocyte antigen (HLA)-G gene were investigated for 201 and 104 healthy unrelated Han samples recruited from Hunan Province, southern China and central Inner Mongolia Autonomous Region, northern China, respectively, using sequence-based typing and cloning methods. Totally 12 HLA-G alleles in the coding region, 9 variable sites in 3'UTR, 8 3'UTR haplotypes and 15 HLA-G extended haplotypes (EHs) incorporating the coding region and 3'UTR were observed. Very strong linkage disequilibrium (LD) was observed between HLA-A and HLA-G, and between HLA-G coding region and 3'UTR in each population (all global P=0.0000). Seven HLA-A-G haplotypes showed significant LD in both populations. Three HLA-G alleles in the coding region, 4 polymorphic sites in the 3'UTR, 3 3'UTR haplotypes and 4 HLA-G EHs differed significantly in their distributions between the 2 Chinese Han populations (all P≤0.0001). There was evidence for balancing selection acting on HLA-G 3'UTR positions +3010, +3142 and +3187 in the two populations. The NJ dendrograms demonstrated the existence of two basic HLA-G lineages and indicated that, HLA-G*01:01:01, the most common HLA-G allele, formed a separate lineage from other alleles. Our results shed new lights into HLA-G genetics among Chinese Han populations. The findings reported here are of importance for future studies related to post-transcriptional regulation of HLA-G allelic expression and the potential role of HLA-G in disease association in populations of Chinese ancestry.

  11. Two lck transcripts containing different 5' untranslated regions are present in T cells.

    PubMed Central

    Voronova, A F; Adler, H T; Sefton, B M

    1987-01-01

    p56lck is a new member of the src family of cellular tyrosine protein kinases. It is expressed constitutively at a low level in normal T cells and at an elevated level in the LSTRA and Thy19 Moloney murine leukemia virus-induced thymoma cell lines. It is possible that the expression of p56lck at an elevated level contributes to the transformation of these thymoma cells. The structure of the mRNAs encoding p56lck was examined by using an RNase protection assay. Both a chimeric lck mRNA containing the 5' untranslated region of Moloney virus mRNA and a normal lck mRNA were found in Thy19 and LSTRA cells. The chimeric lck transcript was 4- to 10-fold more abundant than the normal transcript. Transcription arising from a viral promoter is therefore responsible for the elevated levels of lck mRNA in these two cell lines. Surprisingly, uninfected murine T cells were also found to contain lck transcripts with differing 5' untranslated regions. One species of mRNA was colinear with the region of the chromosome just upstream of the initiation codon for p56lck. The other appeared to arise through splicing of an unidentified 5' untranslated exon to a sometimes cryptic splice acceptor just upstream of the region encoding p56lck. These data suggest that lck is expressed through the use of at least two different promoters. The promoters could be subject to different forms of regulation. Images PMID:3501824

  12. Sdt97: A Point Mutation in the 5' Untranslated Region Confers Semidwarfism in Rice.

    PubMed

    Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

    2016-06-01

    Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5' untranslated region of Sdt97 qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5' untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice.

  13. Sdt97: A Point Mutation in the 5′ Untranslated Region Confers Semidwarfism in Rice

    PubMed Central

    Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

    2016-01-01

    Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5′ untranslated region of Sdt97. qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5′ untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice. PMID:27172200

  14. Sequence analysis of tau 3'untranslated region and saitohin gene in sporadic progressive supranuclear palsy

    PubMed Central

    Ezquerra, M; Campdelacreu, J; Munoz, E; Oliva, R; Tolosa, E

    2004-01-01

    Objectives: To search for genetic changes in the 3'untranslated region (3'UTR) of tau and adjacent sequence LOC147077, and in the coding region of STH in PSP patients. Methods: The study included 57 PSP patients and 83 healthy controls. The genetic analysis of each region was performed through sequencing. The Q7R polymorphism was studied through restriction enzyme and electrophoresis analysis. Results: No mutations were found in the regions analysed. The QQ genotype of the STH polymorphism was over-represented in participants with PSP (91.5%) compared with control subjects (47%) (p⩽0.00001). This genotype co-segregated with the H1/H1 haplotype in our PSP cases. Conclusions: Our results do not support a major role for the tau 3'UTR in PSP genetics. The QQ genotype of STH confers susceptibility for PSP and is in linkage disequilibrium with the H1/H1 haplotype. PMID:14707330

  15. Tissue-specific expression of insulin-like growth factor II mRNAs with distinct 5' untranslated regions

    SciTech Connect

    Irminger, J.C.; Rosen, K.M.; Humble, R.E.; Villa-Komaroff, L.

    1987-09-01

    The authors have used RNA from human hypothalamus as template for the production of cDNAs encoding insulin-like growth factor II (IGF-II). The prohormone coding sequence of brain IGF-II RNA is identical to that found in liver; however, the 5' untranslated sequence of the brain cDNA has no homology to the 5' untranslated sequence of the previously reported liver cDNAs. By using hybridization to specific probes as well as a method based on the properties of RNase H, they found that the human IGF-II gene has at least three exons that encode alternative 5' untranslated regions and that are expressed in a tissue-specific manner. A probe specific to the brain cDNA 5' untranslated region hybridizes to a 6.0-kilobase transcript present in placenta, hypothalamus, adrenal gland, kidney, Wilms tumor, and a pheochromocytoma. The 5' untranslated sequence of the brain cDNA does not hybridize to a 5.3-kilobase transcript found in liver or to a 5.0-kb transcript found in pheochromocytoma. By using RNase H to specifically fragment the IGF-II transcripts into 3' and 5' fragments, they found that the RNAs vary in size due to differences in the 5' end but not the 3' end.

  16. 5' and 3' untranslated regions of pestivirus genome: primary and secondary structure analyses.

    PubMed Central

    Deng, R; Brock, K V

    1993-01-01

    Within the conserved 5' untranslated region (UTR) of the pestivirus genome three highly variable regions were identified. Preceding the polyprotein start codon, multiple cryptic AUG codons and several small open reading frames are characteristic for all the five pestiviruses. Inspection of the context of AUGs revealed that the polyprotein initiation AUG of pestivirus has a weak context for efficient translation initiation. The most favorable context was found in two of the cryptic AUGs. Two oligopyrimidine-rich tracts upstream to the conserved either cryptic or authentic AUG in the 5'-UTR of pestivirus were identified and 83.3% of their nucleotide sequences are complementary to the consensus sequence at the 3' terminus of eucaryotic 18S rRNA. A secondary structure model for the 5'-UTR of pestivirus was predicted. Nucleotide sequence comparison among five pestiviruses led to the identification of a variable region and a conserved region in the 3'-UTR. A deletion of 41 nucleotides was found within the variable region in Osloss. A secondary structure model for the 3'-UTR was also predicted. The structural similarity of the 5'-UTR between pestiviruses and picornaviruses and hepatitis C viruses was demonstrated and the possible implications of features of the 5' and 3'-UTR of pestiviruses are discussed. PMID:8388102

  17. [Association between single nucleotide polymorphisms of 5'-untranslated region of GPx4 gene and male infertility].

    PubMed

    Liu, Shu-yuan; Zhang, Chang-jun; Si, Xiao-min; Yao, Yu-feng; Shi, Lei; Ke, Jin-kun; Yu, Liang; Shi, Li; Yang, Zhao-qin; Huang, Xiao-qin; Sun, Hao; Chu, Jia-you

    2011-06-01

    To study the association between the single nucleotide polymorphisms (SNPs) of the 5'-untranslated region (5'-UTR) of phospholipid hydroperoxide glutathione peroxidase (GPx4 or PHGPx) gene and oligo- or asthenozoospermic male infertility. The 5'-UTR region of the GPx4 gene was amplified from infertile men and controls using the polymerase chain reaction and was analyzed for polymorphisms by direct sequencing. A total of 9 SNPs were present in the cohort, however there were no significant differences in these 9 SNPs between the case and control groups. According to the results of linkage disequilibrium analysis and haplotype construction, one haplotype (rs757229-rs757230-rs4588110-rs3746165-rs3746166: C-G-G-T-A) was present only in the control men, and significant difference was detected(P< 0.01). The SNPs of 5'-UTR region of the GPx4 gene might not be associated with oligo- or asthenozoospermic male infertility. However, the haplotype (rs757229-rs757230-rs4588110- rs3746165-rs3746166: C-G-G-T-A) might be a protective haplotype.

  18. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    ERIC Educational Resources Information Center

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  19. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    ERIC Educational Resources Information Center

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  20. Molecular epidemiology of avian leukosis virus subgroup J and evolutionary history of its 3' untranslated region.

    PubMed

    Zavala, G; Cheng, S; Jackwood, M W

    2007-12-01

    Avian leukosis subgroup J (ALV-J) causes a variety of tumors and mortality in meat-type chickens. Since its discovery in the late 1980s, ALV-J has spread to breeding stock produced by most primary breeding companies of North America, the European Union, and Asia. ALV-J seems to have been eradicated from elite breeding stock produced by most primary breeders, albeit ALV-J still circulates in some commercial poultry. This study was undertaken to examine the molecular epidemiology and evolution of ALV-J detected in breeding stock and broiler chickens representing eight primary breeding companies over a period of approximately 20 yr (1988-2007). The redundant transmembrane region of the envelope gene has been deleted in some isolates, suggesting that this region is dispensable for viral fitness. Within the 3' untranslated region (3' UTR), the direct repeat 1 was present in 100% of the ALV-J isolates studied. In contrast, the E element has undergone substantial deletions in >50% of the ALV-J proviruses studied. Overall, the unique region 3 was the least conserved within the 3' long terminal repeat (LTR), albeit the transcriptional regulatory elements typical of avian retroviruses (CAAT, CArG, PRE, TATA, and Y boxes) were highly conserved. The direct repeat region of the LTR was identical in all of the proviruses, and the 3' unique region 5 was relatively well conserved. Thus, the 3' UTR of ALV-J has evolved rapidly, reflecting significant instability of this region. Some of the mutations in the 3' UTR have resulted in the emergence of moderately distinct genetic lineages representing each primary breeding company from which ALV-J was isolated.

  1. Functions of the 5'- and 3'-untranslated regions of tobamovirus RNA.

    PubMed

    Chujo, Tetsuya; Ishibashi, Kazuhiro; Miyashita, Shuhei; Ishikawa, Masayuki

    2015-08-03

    The tobamovirus genome is a 5'-m(7)G-capped RNA that carries a tRNA-like structure at its 3'-terminus. The genomic RNA serves as the template for both translation and negative-strand RNA synthesis. The 5'- and 3'-untranslated regions (UTRs) of the genomic RNA contain elements that enhance translation, and the 3'-UTR also contains the elements necessary for the initiation of negative-strand RNA synthesis. Recent studies using a cell-free viral RNA translation-replication system revealed that a 70-nucleotide region containing a part of the 5'-UTR is bound cotranslationally by tobacco mosaic virus (TMV) replication proteins translated from the genomic RNA and that the binding leads the genomic RNA to RNA replication pathway. This mechanism explains the cis-preferential replication of TMV by the replication proteins. The binding also inhibits further translation to avoid a fatal ribosome-RNA polymerase collision, which might arise if translation and negative-strand synthesis occur simultaneously on a single genomic RNA molecule. Therefore, the 5'- and 3'-UTRs play multiple important roles in the life cycle of tobamovirus.

  2. Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions.

    PubMed

    Li, Chen; Ge, Ling-ling; Li, Peng-peng; Wang, Yue; Dai, Juan-juan; Sun, Ming-xia; Huang, Li; Shen, Zhi-qiang; Hu, Xiao-chun; Ishag, Hassan; Mao, Xiang

    2014-01-20

    Japanese encephalitis virus is one of the most common causes for epidemic viral encephalitis in humans and animals. Herein we demonstrated that cellular helicase DDX3 is involved in JEV replication. DDX3 knockdown inhibits JEV replication. The helicase activity of DDX3 is crucial for JEV replication. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX3 could interact with JEV non-structural proteins 3 and 5. Co-immunoprecipitation and confocal microscopy analysis confirmed that DDX3 interacts and colocalizes with these viral proteins and viral RNA during the infection. We determined that DDX3 binds to JEV 5' and 3' un-translated regions. We used a JEV-replicon system to demonstrate that DDX3 positively regulates viral RNA translation, which might affect viral RNA replication at the late stage of virus infection. Collectively, we identified that DDX3 is necessary for JEV infection, suggesting that DDX3 might be a novel target to design new antiviral agents against JEV or other flavivirus infections.

  3. Dynamic Regulation of Tandem 3' Untranslated Regions in Zebrafish Spleen Cells during Immune Response.

    PubMed

    Huang, Guangrui; Huang, Shengfeng; Wang, Ruihua; Yan, Xinyu; Li, Yuxin; Feng, Yuchao; Wang, Shaozhou; Yang, Xia; Chen, Liutao; Li, Jun; You, Leiming; Chen, Shangwu; Luo, Guangbin; Xu, Anlong

    2016-01-15

    Alternative polyadenylation (APA) has been found to be involved in tumorigenesis, development, and cell differentiation, as well as in the activation of several subsets of immune cells in vitro. Whether APA takes place in immune responses in vivo is largely unknown. We profiled the variation in tandem 3' untranslated regions (UTRs) in pathogen-challenged zebrafish and identified hundreds of APA genes with ∼ 10% being immune response genes. The detected immune response APA genes were enriched in TLR signaling, apoptosis, and JAK-STAT signaling pathways. A greater number of microRNA target sites and AU-rich elements were found in the extended 3' UTRs than in the common 3' UTRs of these APA genes. Further analysis suggested that microRNA and AU-rich element-mediated posttranscriptional regulation plays an important role in modulating the expression of APA genes. These results indicate that APA is extensively involved in immune responses in vivo, and it may be a potential new paradigm for immune regulation.

  4. Numerical taxonomy of the genus Pestivirus based on palindromic nucleotide substitutions in the 5' untranslated region.

    PubMed

    Giangaspero, Massimo; Harasawa, Ryô

    2007-12-01

    The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5' untranslated region (UTR) of Pestivirus RNA have been considered for taxonomical segregation of species, through the evaluation of 430 genomic sequences. On the basis of qualitative and quantitative secondary structure characteristics, six species have been identified: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), the tentative species Giraffe and a new proposed taxon named Pronghorn. The first step was qualitative and consisted in the characterization of the different positions of the three stems and loops in the 5' UTR sequences of all the strains under consideration belonging to the genus. Secondary structure sequences showing divergent base-pair combinations have been aligned for comparison. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low-variable positions (LVP) including base-pairs present in less than 80% of the genus. The second step was quantitative, allowing the identification of genomic groups by clustering the base-pair combinations according to LVP. Relatedness among types was evaluated to identify homogeneous groups. Cross comparisons between types within the genus have been evaluated by computing the divergence percentage thus clarifying borderline and multirelated sequences.

  5. Translational repression by a miniature inverted-repeat transposable element in the 3′ untranslated region

    PubMed Central

    Shen, Jianqiang; Liu, Juhong; Xie, Kabin; Xing, Feng; Xiong, Fang; Xiao, Jinghua; Li, Xianghua; Xiong, Lizhong

    2017-01-01

    Transposable elements constitute a substantial portion of eukaryotic genomes and contribute to genomic variation, function, and evolution. Miniature inverted-repeat transposable elements (MITEs), as DNA transposons, are widely distributed in plant and animal genomes. Previous studies have suggested that retrotransposons act as translational regulators; however, it remains unknown how host mRNAs are influenced by DNA transposons. Here we report a translational repression mechanism mediated by a stowaway-like MITE (sMITE) embedded in the 3′-untranslated region (3′-UTR) of Ghd2, a member of the CCT (CONSTANS [CO], CO-LIKE and TIMING OF CAB1) gene family in rice. Ghd2 regulates important agronomic traits, including grain number, plant height and heading date. Interestingly, the translational repression of Ghd2 by the sMITE mainly relies on Dicer-like 3a (OsDCL3a). Furthermore, other MITEs in the 3′-UTRs of different rice genes exhibit a similar effect on translational repression, thus suggesting that MITEs may exert a general regulatory function at the translational level. PMID:28256530

  6. The role of RNA structure at 5' untranslated region in microRNA-mediated gene regulation.

    PubMed

    Gu, Wanjun; Xu, Yuming; Xie, Xueying; Wang, Ting; Ko, Jae-Hong; Zhou, Tong

    2014-09-01

    Recent studies have suggested that the secondary structure of the 5' untranslated region (5' UTR) of messenger RNA (mRNA) is important for microRNA (miRNA)-mediated gene regulation in humans. mRNAs that are targeted by miRNA tend to have a higher degree of local secondary structure in their 5' UTR; however, the general role of the 5' UTR in miRNA-mediated gene regulation remains unknown. We systematically surveyed the secondary structure of 5' UTRs in both plant and animal species and found a universal trend of increased mRNA stability near the 5' cap in mRNAs that are regulated by miRNA in animals, but not in plants. Intra-genome comparison showed that gene expression level, GC content of the 5' UTR, number of miRNA target sites, and 5' UTR length may influence mRNA structure near the 5' cap. Our results suggest that the 5' UTR secondary structure performs multiple functions in regulating post-transcriptional processes. Although the local structure immediately upstream of the start codon is involved in translation initiation, RNA structure near the 5' cap site, rather than the structure of the full-length 5' UTR sequences, plays an important role in miRNA-mediated gene regulation. © 2014 Gu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation.

    PubMed

    Bolduc, François; Garant, Jean-Michel; Allard, Félix; Perreault, Jean-Pierre

    2016-10-07

    G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5' and 3'-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed.

  8. Cathepsin C gene 5'-untranslated region mutation in papillon-lefèvre syndrome.

    PubMed

    Kosem, Rok; Debeljak, Maruša; Repič Lampret, Barbka; Kansky, Aleksej; Battelino, Tadej; Trebušak Podkrajšek, Katarina

    2012-01-01

    Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive disorder characterized by palmoplantar keratoderma together with a severe form of generalized aggressive periodontitis and associated with mutations in cathepsin C gene (CTSC). To investigate the clinical and mutational characteristics of 6 PLS patients from 4 unrelated Slovenian families. CTSC mutational and functional analyses were performed. In all patients, a novel homozygous substitution, c.-55C>A, in the CTSC 5'-untranslated region (UTR) was detected on genomic DNA level and confirmed by mRNA analysis, resulting in the almost complete loss of CTSC mRNA expression and CTSC activity. In silico analysis revealed the potential of the mutation to disrupt putative transcription factor binding sites (TFBSs) for AP-2 and Sp families of transcription factors. Identification of a novel CTSC 5'-UTR mutation together with a severe reduction of CTSC mRNA expression and virtually nonexistent CTSC activity was suggestive of a novel mechanism of TFBS dysfunction associated with PLS. Copyright © 2012 S. Karger AG, Basel.

  9. Constitutive translation of human α-synuclein is mediated by the 5'-untranslated region.

    PubMed

    Koukouraki, Pelagia; Doxakis, Epaminondas

    2016-04-01

    Genetic and biochemical studies have established a central role for α-synuclein (SNCA) accumulation in the pathogenesis of Parkinson's disease. Uncovering and subsequently interfering with physiological mechanisms that control SNCA expression is one approach to limit disease progression. To this end, the long and GC-rich 5'-untranslated region (UTR) of SNCA, which is predicted to fold into stable hairpin and G-quadruplex RNA motifs, was investigated for its role in mRNA translation. Inclusion of SNCA 5'-UTR significantly induced expression of both SNCA and luciferase ORF constructs. This effect was not associated with a change in mRNA levels or differential nucleocytoplasmic shuttling. Further, the presence of the 5'-UTR enhanced SNCA synthesis when cap-dependent translation was attenuated with rapamycin treatment. Analysis using multiple methodologies revealed that the 5'-UTR harbours an internal ribosome entry site (IRES) element that spans most of its nucleotide sequence. Signals such as plasma-membrane depolarization, serum starvation and oxidative stress stimulated SNCA protein translation via its 5'-UTR as well as enhanced its IRES activity. Taken together, these data support the idea that the 5'-UTR is an important positive regulator of SNCA synthesis under diverse physiological and pathological conditions, explaining in part the abundance of SNCA in healthy neurons and its accumulation in degenerative cells.

  10. Global changes in processing of mRNA 3' untranslated regions characterize clinically distinct cancer subtypes.

    PubMed

    Singh, Priyam; Alley, Travis L; Wright, Sarah M; Kamdar, Sonya; Schott, William; Wilpan, Robert Y; Mills, Kevin D; Graber, Joel H

    2009-12-15

    Molecular cancer diagnostics are an important clinical advance in cancer management, but new methods are still needed. In this context, gene expression signatures obtained by microarray represent a useful molecular diagnostic. Here, we describe novel probe-level microarray analyses that reveal connections between mRNA processing and neoplasia in multiple tumor types, with diagnostic potential. We now show that characteristic differences in mRNA processing, primarily in the 3'-untranslated region, define molecular signatures that can distinguish similar tumor subtypes with different survival characteristics, with at least 74% accuracy. Using a mouse model of B-cell leukemia/lymphoma, we find that differences in transcript isoform abundance are likely due to both alternative polyadenylation (APA) and differential degradation. While truncation of the 3'-UTR is the most common observed pattern, genes with elongated transcripts were also observed, and distinct groups of affected genes are found in related but distinct tumor types. Genes with elongated transcripts are overrepresented in ontology categories related to cell-cell adhesion and morphology. Analysis of microarray data from human primary tumor samples revealed similar phenomena. Western blot analysis of selected proteins confirms that changes in the 3'-UTR can correlate with changes in protein expression. Our work suggests that alternative mRNA processing, particularly APA, can be a powerful molecular biomarker with prognostic potential. Finally, these findings provide insights into the molecular mechanisms of gene deregulation in tumorigenesis.

  11. RNA-protein interactions of the 3' untranslated regions of myosin heavy chain transcripts.

    PubMed

    Kiri, Arpna; Goldspink, Geoffrey

    2002-01-01

    The RNA-protein interactions of the myosin heavy chain (MyHC) 3' untranslated regions (3'UTRs) were investigated using gel mobility shift assays. Marine skeletal myosin heavy chain mRNAs were amplified using reverse transcription coupled with the polymerase chain reaction (RT-PCR). Four cloned MyHC sequences were identified as slow type 1, fast 2a, fast 2b and fast 2x. The 3'UTRs of the four MyHC mRNAs were shown to interact with muscle protein in a tissue-specific manner as illustrated by gel retardation assays with protein extracts from various tissues. Competition assays indicate that this interaction is specific to the MyHC 3'UTR sequence. UV cross-linking suggests that several small proteins bind to the 3'UTR's. Peptide sequencing identified aldolase A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as MyHC 3'UTR RNA-binding proteins. The implications of these interactions and post-transcriptional regulation of the MyHC genes are discussed.

  12. Identification of short untranslated regions that sufficiently enhance translation in high-quality wheat germ extract.

    PubMed

    Ogawa, Atsushi; Tabuchi, Junichiro; Doi, Yasunori

    2014-08-15

    High-quality wheat germ extract (hqWGE) is very useful for the high-yield production of various types of protein. The most important key to high productivity is the design of mRNA templates. Although the design has been refined for straightforward and efficient translation in hqWGE, there is still room for improvement in untranslated regions (UTRs), especially the 3' UTR length, because a long, cumbersome 3' UTR is commonly used for translation enhancement. Here we examined some short viral 3' cap-independent translation enhancers (3' CITEs) to identify effective ones for efficient translation in hqWGE. We then combined the most effective 3' CITE and a 5' enhancer to further increase the translation efficiency. mRNA with the optimal short 3' and 5' UTRs, both of whose length was less than 150 nt, exhibited a productivity of 1.4 mg/mL in prolonged large-scale protein synthesis in hqWGE, which was comparable to that of control mRNA with a commonly-used long 3' UTR (∼1200 nt).

  13. Constitutive translation of human α-synuclein is mediated by the 5′-untranslated region

    PubMed Central

    Koukouraki, Pelagia; Doxakis, Epaminondas

    2016-01-01

    Genetic and biochemical studies have established a central role for α-synuclein (SNCA) accumulation in the pathogenesis of Parkinson's disease. Uncovering and subsequently interfering with physiological mechanisms that control SNCA expression is one approach to limit disease progression. To this end, the long and GC-rich 5′-untranslated region (UTR) of SNCA, which is predicted to fold into stable hairpin and G-quadruplex RNA motifs, was investigated for its role in mRNA translation. Inclusion of SNCA 5′-UTR significantly induced expression of both SNCA and luciferase ORF constructs. This effect was not associated with a change in mRNA levels or differential nucleocytoplasmic shuttling. Further, the presence of the 5′-UTR enhanced SNCA synthesis when cap-dependent translation was attenuated with rapamycin treatment. Analysis using multiple methodologies revealed that the 5′-UTR harbours an internal ribosome entry site (IRES) element that spans most of its nucleotide sequence. Signals such as plasma-membrane depolarization, serum starvation and oxidative stress stimulated SNCA protein translation via its 5′-UTR as well as enhanced its IRES activity. Taken together, these data support the idea that the 5′-UTR is an important positive regulator of SNCA synthesis under diverse physiological and pathological conditions, explaining in part the abundance of SNCA in healthy neurons and its accumulation in degenerative cells. PMID:27248657

  14. Skipping of an alternative intron in the srsf1 3' untranslated region increases transcript stability.

    PubMed

    Akaike, Yoko; Kurokawa, Ken; Kajita, Keisuke; Kuwano, Yuki; Masuda, Kiyoshi; Nishida, Kensei; Kang, Seung Wan; Tanahashi, Toshihito; Rokutan, Kazuhito

    2011-08-01

    The srsf1 gene encodes serine/arginine-rich splicing factor 1 (SRSF1) that participates in both constitutive and alternative splicing reactions. This gene possesses two ultraconserved elements in the 3' untranslated region (UTR). Skipping of an alternative intron between the two elements has no effect on the protein-coding sequence, but it generates a premature stop codon (PTC)-containing mRNA isoform, whose degradation is considered to depend on nonsense-mediated mRNA decay (NMD). However, several cell lines (HCT116, RKO, HeLa, and WI38 cells) constitutively expressed significant amounts of the srsf1 PTC variant. HCT116 cells expressed the PTC variant nearly equivalent to the major isoform that includes the alternative intron in the 3' UTR. Inhibition of NMD by silencing a key effecter UPF1 or by treatment with cycloheximide failed to increase amounts of the PTC variant in HCT116 cells, and the PTC variant was rather more stable than the major isoform in the presence of actinomycin D. Our results suggest that the original stop codon may escape from the NMD surveillance even in skipping of the alternative intron. The srsf1 gene may produce an alternative splice variant having truncated 3' UTR to relief the microRNA- and/or RNA-binding protein-mediated control of translation or degradation.

  15. Differential regulation of oestrogen receptor β isoforms by 5' untranslated regions in cancer.

    PubMed

    Smith, Laura; Brannan, Rebecca A; Hanby, Andrew M; Shaaban, Abeer M; Verghese, Eldo T; Peter, Mark B; Pollock, Steven; Satheesha, Sampoorna; Szynkiewicz, Marcin; Speirs, Valerie; Hughes, Thomas A

    2010-08-01

    Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs - ERβ- are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5' untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5'UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5'UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function.

  16. Genome-wide profiling of untranslated regions by paired-end ditag sequencing reveals unexpected transcriptome complexity in yeast.

    PubMed

    Kang, Ya-Ni; Lai, Deng-Pan; Ooi, Hong Sain; Shen, Ting-Ting; Kou, Yao; Tian, Jing; Czajkowsky, Daniel M; Shao, Zhifeng; Zhao, Xiaodong

    2015-02-01

    The identification of structural and functional elements encoded in a genome is a challenging task. Although the transcriptome of budding yeast has been extensively analyzed, the boundaries and untranslated regions of yeast genes remain elusive. To address this least-explored field of yeast genomics, we performed a transcript profiling analysis through paired-end ditag (PET) approach coupled with deep sequencing. With 562,133 PET sequences we accurately defined the boundaries and untranslated regions of 3,409 ORFs, suggesting many yeast genes have multiple transcription start sites (TSSs). We also identified 85 previously uncharacterized transcripts either in intergenic regions or from the opposite strand of reported genomic features. Furthermore, our data revealed the extensive 3' end heterogeneity of yeast genes and identified a novel putative motif for polyadenylation. Our results indicate the yeast transcriptome is more complex than expected. This study would serve as an invaluable resource for elucidating the regulation and evolution of yeast genes.

  17. Association of HLA-G 3' untranslated region variants with type 1 diabetes mellitus.

    PubMed

    de Albuquerque, Rafael S; Mendes-Junior, Celso Teixeira; Lucena-Silva, Norma; da Silva, Camila Leal Lopes; Rassi, Diane Meire; Veiga-Castelli, Luciana C; Foss-Freitas, Maria Cristina; Foss, Milton César; Deghaide, Neifi Hassan Saloum; Moreau, Philippe; Gregori, Silvia; Castelli, Erick C; Donadi, Eduardo Antônio

    2016-04-01

    Besides the well recognized association of HLA-DRB1 and DQB1 alleles with type 1 diabetes mellitus (T1D), linkage studies have identified a gene region close to the non-classical class I HLA-G gene as an independent susceptibility marker. HLA-G is constitutively expressed in the endocrine compartment of the human pancreas and may play a role in controlling autoimmune responses. We evaluated the genetic diversity of the 3' untranslated region (3'UTR) of HLA-G, which have been associated with HLA-G mRNA post-transcriptional regulation, in 120 Brazilian T1D patients and in 120 healthy controls. We found the +3001 T allele was observed only in T1D patients. Notably, the +3001 T allele was in linkage disequilibrium with polymorphic sites associated with low production of HLA-G mRNA or soluble HLA-G levels. Moreover, T1D patients showed a low frequency of the HLA-G 3'UTR-17 (14bpINS/+3001T/+3003T/+3010C/+3027C/+3035T/+3142G/+3187A/+3196C). The +3010 CC genotype and the UTR-3 haplotype (14bpDEL/+3001C/+3003T/+3010C/+3027C/+3035C/+3142G/+3187A/+3196C), associated with low and moderate soluble HLA-G expression, respectively, were underrepresented in patients. The decreased expression of HLA-G at the pancreas level should be detrimental in individuals genetically prone to produce less HLA-G.

  18. Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation*

    PubMed Central

    Bolduc, François; Garant, Jean-Michel; Allard, Félix; Perreault, Jean-Pierre

    2016-01-01

    G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5′ and 3′-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo. Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed. PMID:27557661

  19. Phylogenetic study on the 5'-untranslated region of bovine viral diarrhoea virus isolates from Iran.

    PubMed

    Esmaelizad, Majid; Kargar-Moakhar, Rohani

    2014-01-01

    Bovine viral diarrhoea virus is a pathogen of bovids associated with reproduction system, causing in infected animals a range of ailments, from abortion to congenital defects. In this article, the nucleotide structure of the 5'-untranslated region (5-UTR) from 7 Iranian bovine diarrhoea virus (BVDV) isolates was characterized and subjected to comparative analysis against a panel of BVDV isolates from different sources. To this end, a 288 bp-long stretch of the internal ribosome entry site was amplified by RT-PCR. The PCR products subsequently cloned into PTZ57T vector and sequenced using T7 promoter primers. This resulted in detection of 3 new point mutations G → A and G → T in 2 isolates. When these findings were phylogenetically assessed, all the examined Iranian isolates were deemed to belong to the type1 of BVDV. Besides, 2 subtypes were identified among these isolates. In group A, a high level of similarity (99.2%) between Iranian isolates with a cytopathic Australian strain of BVDV-1c was detected; while in group B, the 4 Iranian isolates proved to be very similar to NADL-like BVDV-1a strains. We believe that the surprisingly high level of similarity between group A Iranian isolates and their corresponding Australian strain is likely to be an indication of a shared common ancestor. If correct, the most likely explanation of this observation is the introduction of such strains from Australia to Iran, possibly through exportation of infected live animals or animal productions (e.g. semen and meat) at some points in the past. Nevertheless, this hypothesis remains to be proved as further epidemiological work at genomic level is required to understand population of BVDV in Iran.

  20. Does HIV-1 mRNA 5'-untranslated region bear an internal ribosome entry site?

    PubMed

    Smirnova, Victoria V; Terenin, Ilya M; Khutornenko, Anastasia A; Andreev, Dmitri E; Dmitriev, Sergey E; Shatsky, Ivan N

    2016-02-01

    Unspliced human immunodeficiency virus-1 (HIV-1) mRNA is capped and therefore can be translated via conventional scanning mechanism. In addition, its 5' untranslated region (5'UTR) is thought to function as an internal ribosome entry site (IRES) during G2/M-phase of cell cycle or when cap-dependent translation is inhibited. Recently, customary methods of internal initiation demonstrating have been challenged, and consequently existence of certain IRESs of cellular origin has been put under question. Since a precise knowledge of translation initiation mechanism used by HIV may be important for cure development, presence of the IRES in HIV-1 mRNA demands a careful reexamination using contemporary stringent criteria. The key point of our strategy is to compare translation efficiency of bicistronic mRNA bearing HIV-1 unspliced mRNA 5' UTR in the intercistronic position to that of the corresponding capped monocistronic mRNA. This approach allows determination of internal initiation contribution into the overall level of particular mRNA translation. We found that both in cell-free systems and in cultured cells monocistronic mRNA with HIV-1 unspliced mRNA 5'UTR is translated significantly better than bicistronic one. Importantly, it is also true for G2/M-phase stalled cells or for cells under conditions of inhibited cap-dependent translation. Thus, in our hands contribution of internal ribosome entry into the overall level of translation driven by HIV-1 unspliced mRNA 5'UTR is negligible, and 5'-dependent scanning is a primary mechanism of its translation initiation.

  1. Upstream open reading frame in 5'-untranslated region reduces titin mRNA translational efficiency.

    PubMed

    Cadar, Adrian G; Zhong, Lin; Lin, Angel; Valenzuela, Mauricio O; Lim, Chee C

    2014-10-10

    Titin is the largest known protein and a critical determinant of myofibril elasticity and sarcomere structure in striated muscle. Accumulating evidence that mRNA transcripts are post-transcriptionally regulated by specific motifs located in the flanking untranslated regions (UTRs) led us to consider the role of titin 5'-UTR in regulating its translational efficiency. Titin 5'-UTR is highly homologous between human, mouse, and rat, and sequence analysis revealed the presence of a stem-loop and two upstream AUG codons (uAUGs) converging on a shared in frame stop codon. We generated a mouse titin 5'-UTR luciferase reporter construct and targeted the stem-loop and each uAUG for mutation. The wild-type and mutated constructs were transfected into the cardiac HL-1 cell line and primary neonatal rat ventricular myocytes (NRVM). SV40 driven 5'-UTR luciferase activity was significantly suppressed by wild-type titin 5'-UTR (∼ 70% in HL-1 cells and ∼ 60% in NRVM). Mutating both uAUGs was found to alleviate titin 5'-UTR suppression, while eliminating the stem-loop had no effect. Treatment with various growth stimuli: pacing, PMA or neuregulin had no effect on titin 5'-UTR luciferase activity. Doxorubicin stress stimuli reduced titin 5'-UTR suppression, while H2O2 had no effect. A reported single nucleotide polymorphism (SNP) rs13422986 at position -4 of the uAUG2 was introduced and found to further repress titin 5'-UTR luciferase activity. We conclude that the uAUG motifs in titin 5'-UTR serve as translational repressors in the control of titin gene expression, and that mutations/SNPs of the uAUGs or doxorubicin stress could alter titin translational efficiency.

  2. 5' and 3' untranslated regions contribute to the differential expression of specific HLA-A alleles.

    PubMed

    René, Céline; Lozano, Claire; Villalba, Martin; Eliaou, Jean-François

    2015-12-01

    In hematopoietic stem cell transplantation (HSCT), when no HLA full-matched donor is available, alternative donors could include one HLA-mismatched donor. Recently, the low expressed HLA-C alleles have been identified as permissive mismatches for the best donor choice. Concerning HLA-A, the degree of variability of expression is poorly understood. Here, we evaluated HLA-A expression in healthy individuals carrying HLA-A*02 allele in different genotypes using flow cytometry and allele-specific quantitative RT-PCR. While an interindividual variability of HLA-A*02 cell surface expression, not due to the allele associated, was observed, no difference of the mRNA expression level was shown, suggesting the involvement of the posttranscriptional regulation. The results of qRT-PCR analyses exhibit a differential expression of HLA-A alleles with HLA-A*02 as the strongest expressed allele independently of the second allele. The associated non-HLA-A*02 alleles were differentially expressed, particularly the HLA-A*31 and HLA-A*33 alleles (strong expression) and the HLA-A*29 (low expression). The presence of specific polymorphisms in the 5' and 3' untranslated regions of the HLA-A*31 and HLA-A*33 alleles could contribute to this high level of expression. As previously described for HLA-C, low-expressed HLA-A alleles, such as HLA-A*29, could be considered as a permissive mismatch, although this needs to be confirmed by clinical studies.

  3. Feeding and insulin increase leptin translation. Importance of the leptin mRNA untranslated regions.

    PubMed

    Lee, Mi-Jeong; Yang, Rong-Ze; Gong, Da-Wei; Fried, Susan K

    2007-01-05

    The post-transcriptional mechanisms by which feeding and insulin increase leptin production are poorly understood. Starvation of 6-7-week-old rats for 14 h decreased leptin mRNA level by only 22% but decreased plasma levels, adipose tissue leptin content, and release by over 75%. The decreased leptin with starvation was explained by >85% decrease in relative rates of leptin biosynthesis measured by metabolic labeling and immunoprecipitation. In vitro insulin treatment of adipose tissue from fed or starved rats for 2 h increased relative rates of leptin biosynthesis by 2-3-fold, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Consistent with the hypothesis that feeding/insulin increases leptin translation, more leptin mRNA was associated with polysomes in adipose tissue of fed than starved rats, and in vitro incubation of adipose tissue of starved rats with insulin shifted leptin mRNA into polysomes. To assess the mechanisms regulating leptin translation, chimeric human leptin untranslated region (UTR) reporter constructs were transiently transfected into differentiated 3T3-L1 adipocytes. The 5'-UTR of leptin mRNA increased luciferase reporter activity 2-3-fold, whereas the full-length 3'-UTR (nucleotides 1-2804) was inhibitory (-65%). Sequences between nucleotides 462 and 1130 of the leptin 3'-UTR conferred most of the inhibitory effect. Insulin stimulated the expression of constructs that included both the full-length 5'-UTR and the inhibitory 3'-UTR, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Our data suggest that insulin derepresses leptin translation by a mechanism that requires both the 5'-UTR and the 3'-UTR and may contribute to the increase in leptin production with feeding.

  4. MicroRNA genes and their target 3'-untranslated regions are infrequently somatically mutated in ovarian cancers.

    PubMed

    Ryland, Georgina L; Bearfoot, Jennifer L; Doyle, Maria A; Boyle, Samantha E; Choong, David Y H; Rowley, Simone M; Tothill, Richard W; Gorringe, Kylie L; Campbell, Ian G

    2012-01-01

    MicroRNAs are key regulators of gene expression and have been shown to have altered expression in a variety of cancer types, including epithelial ovarian cancer. MiRNA function is most often achieved through binding to the 3'-untranslated region of the target protein coding gene. Mutation screening using massively-parallel sequencing of 712 miRNA genes in 86 ovarian cancer cases identified only 5 mutated miRNA genes, each in a different case. One mutation was located in the mature miRNA, and three mutations were predicted to alter the secondary structure of the miRNA transcript. Screening of the 3'-untranslated region of 18 candidate cancer genes identified one mutation in each of AKT2, EGFR, ERRB2 and CTNNB1. The functional effect of these mutations is unclear, as expression data available for AKT2 and EGFR showed no increase in gene transcript. Mutations in miRNA genes and 3'-untranslated regions are thus uncommon in ovarian cancer.

  5. MicroRNA Genes and Their Target 3′-Untranslated Regions Are Infrequently Somatically Mutated in Ovarian Cancers

    PubMed Central

    Doyle, Maria A.; Boyle, Samantha E.; Choong, David Y. H.; Rowley, Simone M.; Tothill, Richard W.; Gorringe, Kylie L.; Campbell, Ian G.

    2012-01-01

    MicroRNAs are key regulators of gene expression and have been shown to have altered expression in a variety of cancer types, including epithelial ovarian cancer. MiRNA function is most often achieved through binding to the 3′-untranslated region of the target protein coding gene. Mutation screening using massively-parallel sequencing of 712 miRNA genes in 86 ovarian cancer cases identified only 5 mutated miRNA genes, each in a different case. One mutation was located in the mature miRNA, and three mutations were predicted to alter the secondary structure of the miRNA transcript. Screening of the 3′-untranslated region of 18 candidate cancer genes identified one mutation in each of AKT2, EGFR, ERRB2 and CTNNB1. The functional effect of these mutations is unclear, as expression data available for AKT2 and EGFR showed no increase in gene transcript. Mutations in miRNA genes and 3′-untranslated regions are thus uncommon in ovarian cancer. PMID:22536442

  6. Species characterization in the genus Pestivirus according to palindromic nucleotide substitutions in the 5'-untranslated region.

    PubMed

    Giangaspero, Massimo; Harasawa, Ryô

    2011-06-01

    The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5'-untranslated region (UTR) of the Pestivirus genome have been considered for taxonomical segregation of the species, through the evaluation of 534 strains. On the basis of qualitative and quantitative secondary structure characteristics, species have been identified within the genus, determining genetic distances between species isolates, clarifying borderline and multirelated sequences, and characterizing and clustering the Pestivirus strains showing unexpected genomic sequences. Nine genomic groups have been identified: the species Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV) and the tentative species Pronghorn, Giraffe, Bovine viral diarrhea virus 3 (BVDV-3) (HoBi group), Border disease virus 2 (BDV-2) (Italian small ruminant isolates) and Bungowannah. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low variable positions (LVP) including base-pairs present in less than 80% of the genus. The determination of divergence between single strain sequences or genetic groups was obtained easily by comparing base-pairing combinations from aligned secondary structures. This provided clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. The BVDV-1 and BDV species resulted heterogeneous, showing isolates located on a borderline in the species. Within the BVDV-2 species, two main genogroups were identified, with strains showing common sequence characteristics to both groups (multirelated strains). They could be allocated correctly by quantitative analysis. Similarly, the relation between CSFV and BDV species appeared very clearly. Also in this case, ambiguous strain sequences could be clustered in the

  7. Regions in the 3' untranslated region confer stage-specific expression to the Leishmania mexicana a600-4 gene.

    PubMed

    Murray, Angus; Fu, Christine; Habibi, Golareh; McMaster, W Robert

    2007-06-01

    Protozoan parasites of the genus Leishmania have a digenetic lifecycle, alternating between the promastigote and amastigote stages. The extracellular promastigote resides within a sandfly vector, while the obligate intracellular amastigote stage replicates in the phagolysosome of mammalian host macrophages. Adaptation to and survival within these vastly differently environments is accompanied by differential expression of a subset of genes, which is regulated post-transcriptionally via cis-acting elements in 3' untranslated region (3'UTR) or intercistronic sequences. It was reported previously that Leishmania mexicana A600-4 mRNA transcript abundance was eight-fold higher in the amastigotes. In this study, chimeric luciferase:A600-4 3'UTR reporter constructs were integrated at the A600 chromosome locus to identify regulatory regions of the A600-4 3'UTR sequence. Evidence is provided for distinct 3'UTR elements that function to stabilize the A600-4 mRNA transcript in the amastigote stage and to regulate translation efficiency, respectively.

  8. Molecular characterization of enterovirus 71 and coxsackievirus A16 using the 5' untranslated region and VP1 region.

    PubMed

    Zhou, Fei; Kong, Fanrong; Wang, Bin; McPhie, Kenneth; Gilbert, Gwendolyn L; Dwyer, Dominic E

    2011-03-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the species Human enterovirus A, and are both major and independent aetiological agents of hand-foot-and-mouth disease. The human enterovirus (HEV) 5' untranslated region (UTR) is fundamentally important for efficient virus replication and for virulence, whilst the VP1 region correlates well with antigenic typing by neutralization, and can be used for virus identification and evolutionary studies. A comparison was undertaken of the 5'UTR and VP1 nucleotide sequences of five EV71 clinical isolates and 10 CVA16 clinical isolates from one laboratory with the 5'UTR and VP1 sequences of 104 EV71 strains and 45 CVA16 strains available in GenBank. The genetic relationships were analysed using standard phylogenetic methods. The EV71 phylogenetic analysis showed that the VP1 sequences were clustered into three genogroups, A, B and C, with genogroups B and C further divided into five subgenogroups, B1-B5 and C1-C5, respectively. All EV71 strains were clustered similarly in the 5'UTR and VP1 trees, except for one Taiwanese strain, which demonstrated different clustering in the two trees, suggesting a recombination event in the phylogeny. The CVA16 phylogenetic analysis showed that the VP1 sequences were clustered into two genogroups, A and B, with genogroup B further divided into B1 (B1a and B1b), B2 and a possible B3; and that a similar pattern and grouping of all strains were displayed in the 5'UTR tree. This study demonstrated that comparing the two regions provides evidence of epidemiological linkage of HEV-A strains, and that mutation in the two regions plays a vital role in the evolution of these viruses. The combination of molecular typing and phylogenetic sequence analysis will be beneficial in both individual patient diagnosis and public health measures.

  9. A guide to design and optimization of reporter assays for 3' untranslated region mediated regulation of mammalian messenger RNAs.

    PubMed

    Van Etten, Jamie; Schagat, Trista L; Goldstrohm, Aaron C

    2013-09-15

    Post-transcriptional regulatory mechanisms are pervasive in the control of gene expression. Regulatory sequences within transcripts can control RNA processing, localization, translation efficiency, and stability of the RNA. Regulation is mediated by a diverse set of RNA binding regulators, including proteins and RNAs, which interact with specific mRNA sequences that are often found in untranslated regions. The potential for vast post-transcriptional control exists: mammalian mRNAs contain extensive untranslated regions and their genomes encode many hundreds of RNA binding proteins and non-coding RNAs. Facile quantitative methods are necessary to study the activities and mechanisms of regulatory sequences and the RNA binding factors that recognize them. Here we discuss the design and implementation of luciferase-based reporter assays to measure the effect of regulatory RNA sequences on protein and RNA expression. Protocols are described for transfection of the reporter into cells, measurement of protein expression levels with luciferase activity assays, RNA purification, and measurement of mRNA levels by reverse-transcription and quantitative polymerase chain reaction. For each assay, troubleshooting of common problems and critical controls are discussed. We present our optimized techniques and data from studies that measure specific and direct repression (i.e. negative regulation) of mRNAs by members of the PUF family of RNA binding proteins in cultured human cells.

  10. Loop structures in the 5' untranslated region and antisense RNA mediate pilE gene expression in Neisseria gonorrhoeae.

    PubMed

    Masters, Thao L; Wachter, Jenny; Hill, Stuart A

    2016-11-01

    Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.

  11. 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.

  12. Incorporation of beta-globin untranslated regions into a Sindbis virus vector for augmentation of heterologous mRNA expression.

    PubMed

    Strong, T V; Hampton, T A; Louro, I; Bilbao, G; Conry, R M; Curiel, D T

    1997-06-01

    Polynucleotide immunization has been employed as a means of inducing immune responses through the introduction of antigen-encoding DNA. While immunization against specific tumor antigens may be achieved through this strategy, various candidate tumor antigens may not be approached via DNA-based vaccines as they represent transforming oncogenes. As an alternative approach, we have explored the utility of mRNA vectors for polynucleotide immunization. The transient expression achieved by mRNA may provide an efficient and safe system for stimulating immune responses to tumor-specific antigens. Our previous work demonstrated that a self-replicating RNA enhances the magnitude and duration of transgene expression for this application. Here we further modify the vector for optimal use in gene therapy through the incorporation of untranslated regions flanking the encoded transgene. The beta-globin 5' and 3' untranslated regions (UTRs) were inserted directly flanking the luciferase gene in both nonreplicative and replicative RNA constructs. In both cases, elevated and prolonged levels of luciferase expression were detected from the beta-globin UTR-flanked luciferase as compared to luciferase without these sequences. These modifications improve the ability of replicative RNA vectors to produce high, yet transient transgene expression for cancer immunotherapy strategies.

  13. Absence of association between a polymorphic GGC repeat in the 5' untranslated region of the reelin gene and autism.

    PubMed

    Krebs, M O; Betancur, C; Leroy, S; Bourdel, M C; Gillberg, C; Leboyer, M

    2002-01-01

    Autism is a complex neurodevelopmental disorder with severe cognitive and communication disabilities, that has a strong genetic predisposition. Reelin, a protein involved in neuronal migration during development, is encoded by a gene located on 7q22, within the candidate region on 7q showing increased allele sharing in previous genome scans. A case/control and family-based association study recently reported a positive association between a trinucleotide repeat polymorphism (GGC) located in the 5' untranslated region (UTR) of the reelin gene and autism. We performed a transmission disequilibrium test (TDT) analysis of the 5'UTR polymorphism in 167 families including 218 affected subjects (117 trios and 50 affected sib pairs) and found no evidence of linkage/association. Our results do not support previous findings and suggest that this GGC polymorphism of the reelin gene is unlikely to be a major susceptibility factor in autism and/or genetic heterogeneity.

  14. Absence of association of a polymorphic GGC repeat at the 5' untranslated region of the reelin gene with schizophrenia.

    PubMed

    Huang, Chia-Hsing; Chen, Chia-Hsiang

    2006-05-30

    Reelin is an extracellular matrix glycoprotein that plays an important role in guiding neuronal migration, lamination and connection during embryonic brain development. Several reports suggest that reduced reelin expression is associated with human mental illnesses such as schizophrenia, mood disorders and autism. Human reelin cDNA has been cloned and contains a polymorphic GGC repeat at the 5' untranslated region. In view of the possible regulation of reelin gene expression by this GGC polymorphism, we investigated the association of the polymorphic GGC repeat with schizophrenia in a Chinese Han population from Taiwan. We found no differences of allelic and genotypic distributions of the polymorphic GGC triplets between 162 schizophrenic patients and 176 controls in this study. Our findings do not support the involvement of the polymorphic GGC triplets of the reelin gene in the pathogenesis of schizophrenia in the population studied.

  15. Dopa-responsive dystonia: functional analysis of single nucleotide substitutions within the 5' untranslated GCH1 region.

    PubMed

    Armata, Ioanna A; Balaj, Leonora; Kuster, John K; Zhang, Xuan; Tsai, Shelun; Armatas, Andreas A; Multhaupt-Buell, Trisha J; Soberman, Roy; Breakefield, Xandra O; Ichinose, Hiroshi; Sharma, Nutan

    2013-01-01

    Mutations in the GCH1 gene are associated with childhood onset, dopa-responsive dystonia (DRD). Correct diagnosis of DRD is crucial, given the potential for complete recovery once treated with L-dopa. The majority of DRD associated mutations lie within the coding region of the GCH1 gene, but three additional single nucleotide sequence substitutions have been reported within the 5' untranslated (5'UTR) region of the mRNA. The biologic significance of these 5'UTR GCH1 sequence substitutions has not been analyzed. Luciferase reporter assays, quantitative real time PCR and RNA decay assays, combined with bioinformatics, revealed a pathogenic 5'UTR GCH1 substitution. The +142C>T single nucleotide 5'UTR substitution that segregates with affected status in DRD patients, substantially attenuates translation without altering RNA expression levels or stability. The +142C>T substitution disrupts translation most likely by creating an upstream initiation start codon (uAUG) and an upstream open reading frame (uORF). This is the first GCH1 regulatory substitution reported to act at a post-transcriptional level, increasing the list of genetic diseases caused by abnormal translation and reaffirming the importance of investigating potential regulatory substitutions in genetic diseases.

  16. Absence of association between a polymorphic GGC repeat in the 5' untranslated region of the reelin gene and autism

    PubMed Central

    Krebs, Marie-Odile; Betancur, Catalina; Leroy, Sophie; Bourdel, Marie-Chantal; Gillberg, Christopher; Leboyer, Marion

    2002-01-01

    Autism is a complex neurodevelopmental disorder with severe cognitive and communication disabilities, that has a strong genetic predisposition predisposition.1 Reelin, a protein involved in neuronal migration during development, is encoded by a gene located on 7q22, 7q22,2 within the candidate region on 7q showing increased allele sharing in previous genome scans. 3–8 A case/control and family-based association study recently reported a positive association between a trinucleotide repeat polymorphism (GGC) located in the 5′ untranslated region (UTR) of the reelin gene and autism. 9 We performed a transmission disequilibrium test (TDT) analysis of the 5′UTR polymorphism in 167 families including 218 affected subjects (117 trios and 50 affected sib pairs) and found no evidence of linkage/association. Our results do not support previous findings and suggest that the reelin gene is unlikely to play a major role as a susceptibility factor in autism and/or genetic heterogeneity. PMID:12192627

  17. Computational identification of new structured cis-regulatory elements in the 3'-untranslated region of human protein coding genes.

    PubMed

    Chen, Xiaowei Sylvia; Brown, Chris M

    2012-10-01

    Messenger ribonucleic acids (RNAs) contain a large number of cis-regulatory RNA elements that function in many types of post-transcriptional regulation. These cis-regulatory elements are often characterized by conserved structures and/or sequences. Although some classes are well known, given the wide range of RNA-interacting proteins in eukaryotes, it is likely that many new classes of cis-regulatory elements are yet to be discovered. An approach to this is to use computational methods that have the advantage of analysing genomic data, particularly comparative data on a large scale. In this study, a set of structural discovery algorithms was applied followed by support vector machine (SVM) classification. We trained a new classification model (CisRNA-SVM) on a set of known structured cis-regulatory elements from 3'-untranslated regions (UTRs) and successfully distinguished these and groups of cis-regulatory elements not been strained on from control genomic and shuffled sequences. The new method outperformed previous methods in classification of cis-regulatory RNA elements. This model was then used to predict new elements from cross-species conserved regions of human 3'-UTRs. Clustering of these elements identified new classes of potential cis-regulatory elements. The model, training and testing sets and novel human predictions are available at: http://mRNA.otago.ac.nz/CisRNA-SVM.

  18. The BCL-2 5′ Untranslated Region Contains an RNA G-Quadruplex-Forming Motif That Modulates Protein Expression†

    PubMed Central

    Shahid, Ramla; Bugaut, Anthony; Balasubramanian, Shankar

    2011-01-01

    The BCL-2 gene encodes a 25 kDa membrane protein that plays critical roles in the control of apoptosis. The regulation of BCL-2 gene expression is highly complex and occurs both transcriptionally and posttranscriptionally. In particular, the 5′ upstream region of BCL-2 contains a number of elements that control its expression. We have identified a highly conserved 25-nucleotide G-rich sequence (BCL2Q), with potential to fold into a RNA G-quadruplex structure, located 42 nucleotides upstream of the translation start site of human BCL-2. In this study, we used a series of biophysical experiments to show that the BCL2Q sequence folds into a stable RNA G-quadruplex in vitro, and we conducted functional luciferase reporter-based assays, in a cell-free lysate and in three types of human cell lines, to demonstrate that the BCL2Q sequence modulates protein expression in the context of the 493-nucleotide native 5′ untranslated region of BCL-2. PMID:20726580

  19. Extensive sequence variation in the 3′ untranslated region of the KRAS gene in lung and ovarian cancer cases

    PubMed Central

    Kim, Minlee; Chen, Xiaowei; Chin, Lena J; Paranjape, Trupti; Speed, William C; Kidd, Kenneth K; Zhao, Hongyu; Weidhaas, Joanne B; Slack, Frank J

    2014-01-01

    While cancer is a serious health issue, there are very few genetic biomarkers that predict predisposition, prognosis, diagnosis, and treatment response. Recently, sequence variations that disrupt microRNA (miRNA)-mediated regulation of genes have been shown to be associated with many human diseases, including cancer. In an early example, a variant at one particular single nucleotide polymorphism (SNP) in a let-7 miRNA complementary site in the 3′ untranslated region (3′ UTR) of the KRAS gene was associated with risk and outcome of various cancers. The KRAS oncogene is an important regulator of cellular proliferation, and is frequently mutated in cancers. To discover additional sequence variants in the 3′ UTR of KRAS with the potential as genetic biomarkers, we resequenced the complete region of the 3′ UTR of KRAS in multiple non-small cell lung cancer and epithelial ovarian cancer cases either by Sanger sequencing or capture enrichment followed by high-throughput sequencing. Here we report a comprehensive list of sequence variations identified in cases, with some potentially dysregulating expression of KRAS by altering putative miRNA complementary sites. Notably, rs712, rs9266, and one novel variant may have a functional role in regulation of KRAS by disrupting complementary sites of various miRNAs, including let-7 and miR-181. PMID:24552817

  20. Distribution of mutational fitness effects and of epistasis in the 5' untranslated region of a plant RNA virus.

    PubMed

    Bernet, Guillermo P; Elena, Santiago F

    2015-12-07

    Understanding the causes and consequences of phenotypic variability is a central topic of evolutionary biology. Mutations within non-coding cis-regulatory regions are thought to be of major effect since they affect the expression of downstream genes. To address the evolutionary potential of mutations affecting such regions in RNA viruses, we explored the fitness properties of mutations affecting the 5'-untranslated region (UTR) of a prototypical member of the picorna-like superfamily, Tobacco etch virus (TEV). This 5' UTR acts as an internal ribosomal entry site (IRES) and is essential for expression of all viral genes. We determined in vitro the folding of 5' UTR using the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) technique. Then, we created a collection of single-nucleotide substitutions on this region and evaluated the statistical properties of their fitness effects in vivo. We found that, compared to random mutations affecting coding sequences, mutations at the 5' UTR were of weaker effect. We also created double mutants by combining pairs of these single mutations and found variation in the magnitude and sign of epistatic interactions, with an enrichment of cases of positive epistasis. A correlation exists between the magnitude of fitness effects and the size of the perturbation made in the RNA folding structure, suggesting that the larger the departure from the predicted fold, the more negative impact in viral fitness. Evidence that mutational fitness effects on the short 5' UTR regulatory sequence of TEV are weaker than those affecting its coding sequences have been found. Epistasis among pairs of mutations on the 5' UTR ranged between the extreme cases of synthetic lethal and compensatory. A plausible hypothesis to explain all these observations is that the interaction between the 5' UTR and the host translational machinery was shaped by natural selection to be robust to mutations, thus ensuring the homeostatic expression of viral

  1. Distinct 3'-untranslated region elements regulate stage-specific mRNA accumulation and translation in Leishmania.

    PubMed

    McNicoll, François; Müller, Michaela; Cloutier, Serge; Boilard, Nathalie; Rochette, Annie; Dubé, Marthe; Papadopoulou, Barbara

    2005-10-21

    We recently characterized a large developmentally regulated gene family in Leishmania encoding the amastin surface proteins. While studying the regulation of these genes, we identified a region of 770 nucleotides (nt) within the 2055-nt 3'-untranslated region (3'-UTR) that regulates stage-specific gene expression at the level of translation. An intriguing feature of this 3'-UTR regulatory region is the presence of a approximately 450-nt element that is highly conserved among several Leishmania mRNAs. Here we show, using a luciferase reporter system and polysome profiling experiments, that the 450-nt element stimulates translation initiation of the amastin mRNA in response to heat shock, which is the main environmental change that the parasite encounters upon its entry into the mammalian host. Deletional analyses depicted a second region of approximately 100 nucleotides located at the 3'-end of several amastin transcripts, which also activates translation in response to elevated temperature. Both 3'-UTR regulatory elements act in an additive manner to stimulate amastin mRNA translation. In addition, we show that acidic pH encountered in the phagolysosomes of macrophages, the location of parasitic differentiation, triggers the accumulation of amastin transcripts by a distinct mechanism that is independent of the 450-nt and 100-nt elements. Overall, these important findings support the notion that stage-specific post-transcriptional regulation of the amastin mRNAs in Leishmania is complex and involves the coordination of distinct mechanisms controlling mRNA stability and translation that are independently triggered by key environmental signals inducing differentiation of the parasite within macrophages.

  2. Endothelin-1 expression is strongly repressed by AU-rich elements in the 3'-untranslated region of the gene.

    PubMed

    Reimunde, Francisco M; Castañares, Cristina; Redondo-Horcajo, Mariano; Lamas, Santiago; Rodríguez-Pascual, Fernando

    2005-05-01

    The regulation of the synthesis of the endothelial-derived vasoconstrictor ET-1 (endothelin-1) is a complex process that occurs mainly at the mRNA level. Transcription of the gene accounts for an important part of the regulation of expression, as already described for different modulators such as the cytokine TGF-beta (transforming growth factor-beta). However, very little is known about mechanisms governing ET-1 expression at the post-transcriptional level. The aim of the present study was to investigate the regulation of the ET-1 expression at this level. Since the 3'-UTR (3'-untranslated region) of mRNAs commonly contains genetic determinants for the post-transcriptional control of gene expression, we focused on the potential role of the 3'-UTR of ET-1 mRNA. Experiments performed with luciferase reporter constructs containing the 3'-UTR showed that this region exerts a potent destabilizing effect. Deletional analyses allowed us to locate this activity within a region at positions 924-1127. Some (but not all) of the AREs (AU-rich elements) present in this region were found to be essential for this mRNA-destabilizing activity. We also present evidence that cytosolic proteins from endothelial cells interact specifically with these RNA elements, and that a close correlation exists between the ability of the AREs to destabilize ET-1 mRNA and the binding of proteins to these elements. Our results are compatible with the existence of a strong repressional control of ET-1 expression mediated by destabilization of the mRNA exerted through the interaction of specific cytosolic proteins with AREs present in the 3'-UTR of the gene.

  3. Untranslated regions of mRNA and their role in regulation of gene expression in protozoan parasites.

    PubMed

    Rao, Shilpa J; Chatterjee, Sangeeta; Pal, Jayantapal K

    2017-03-01

    Protozoan parasites are one of the oldest living entities in this world that throughout their existence have shown excellent resilience to the odds of survival and have adapted beautifully to ever changing rigors of the environment. In view of the dynamic environment encountered by them throughout their life cycle, and in establishing pathogenesis, it is unsurprising that modulation of gene expression plays a fundamental role in their survival. In higher eukaryotes, untranslated regions (UTRs) of transcripts are one of the crucial regulators of gene expression (influencing mRNA stability and translation efficiency). Parasitic protozoan genome studies have led to the characterization (in silico, in vitro and in vivo) of a large number of their genes. Comparison of higher eukaryotic UTRs with parasitic protozoan UTRs reveals the existence of several similar and dissimilar facets of the UTRs. This review focuses on the elements of UTRs of medically important protozoan parasites and their regulatory role in gene expression. Such information may be useful to researchers in designing gene targeting strategies linked with perturbation of host-parasite relationships leading to control of specific parasites.

  4. The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation.

    PubMed

    Vallejos, Maricarmen; Ramdohr, Pablo; Valiente-Echeverría, Fernando; Tapia, Karla; Rodriguez, Felipe E; Lowy, Fernando; Huidobro-Toro, J Pablo; Dangerfield, John A; López-Lastra, Marcelo

    2010-01-01

    In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

  5. The G-quadruplex augments translation in the 5' untranslated region of transforming growth factor β2.

    PubMed

    Agarwala, Prachi; Pandey, Satyaprakash; Mapa, Koyeli; Maiti, Souvik

    2013-03-05

    Transforming growth factor β2 (TGFβ2) is a versatile cytokine with a prominent role in cell migration, invasion, cellular development, and immunomodulation. TGFβ2 promotes the malignancy of tumors by inducing epithelial-mesenchymal transition, angiogenesis, and immunosuppression. As it is well-documented that nucleic acid secondary structure can regulate gene expression, we assessed whether any secondary motif regulates its expression at the post-transcriptional level. Bioinformatics analysis predicts an existence of a 23-nucleotide putative G-quadruplex sequence (PG4) in the 5' untranslated region (UTR) of TGFβ2 mRNA. The ability of this stretch of sequence to form a highly stable, intramolecular parallel quadruplex was demonstrated using ultraviolet and circular dichroism spectroscopy. Footprinting studies further validated its existence in the presence of a neighboring nucleotide sequence. Following structural characterization, we evaluated the biological relevance of this secondary motif using a dual luciferase assay. Although PG4 inhibits the expression of the reporter gene, its presence in the context of the entire 5' UTR sequence interestingly enhances gene expression. Mutation or removal of the G-quadruplex sequence from the 5' UTR of the gene diminished the level of expression of this gene at the translational level. Thus, here we highlight an activating role of the G-quadruplex in modulating gene expression of TGFβ2 at the translational level and its potential to be used as a target for the development of therapeutics against cancer.

  6. The WOR 1 5′ untranslated region regulates white‐opaque switching in C andida albicans by reducing translational efficiency

    PubMed Central

    Guan, Zhiyun

    2015-01-01

    Summary The human fungal pathogen C andida albicans undergoes white‐opaque phenotypic switching, which enhances its adaptation to host niches. Switching is controlled by a transcriptional regulatory network of interlocking feedback loops acting on the transcription of WOR 1, the master regulator of white‐opaque switching, but regulation of the network on the translational level is not yet explored. Here, we show that the long 5′ untranslated region of WOR 1 regulates the white‐opaque phenotype. Deletion of the WOR 1 5′ UTR promotes white‐to‐opaque switching and stabilizes the opaque state. The WOR 1 5′ UTR reduces translational efficiency and the association of the transcript with polysomes. Reduced polysome association was observed for additional key regulators of cell fate and morphology with long 5′ UTR as well. Overall, we find a novel regulatory step of white‐opaque switching at the translational level. This translational regulation is implicated for many key regulators of cell fate and morphology in C . albicans. PMID:25831958

  7. Translational control of the Xenopus laevis connexin-41 5'-untranslated region by three upstream open reading frames.

    PubMed

    Meijer, H A; Dictus, W J; Keuning, E D; Thomas, A A

    2000-10-06

    The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5'-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5'-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stage Xenopus embryos. The translational efficiency of the wild-type Cx41 5'-UTR was only 2% compared with that of the beta-globin 5'-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5'-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5'-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency in Xenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.

  8. The WOR1 5' untranslated region regulates white-opaque switching in Candida albicans by reducing translational efficiency.

    PubMed

    Guan, Zhiyun; Liu, Haoping

    2015-07-01

    The human fungal pathogen Candida albicans undergoes white-opaque phenotypic switching, which enhances its adaptation to host niches. Switching is controlled by a transcriptional regulatory network of interlocking feedback loops acting on the transcription of WOR1, the master regulator of white-opaque switching, but regulation of the network on the translational level is not yet explored. Here, we show that the long 5' untranslated region of WOR1 regulates the white-opaque phenotype. Deletion of the WOR1 5' UTR promotes white-to-opaque switching and stabilizes the opaque state. The WOR1 5' UTR reduces translational efficiency and the association of the transcript with polysomes. Reduced polysome association was observed for additional key regulators of cell fate and morphology with long 5' UTR as well. Overall, we find a novel regulatory step of white-opaque switching at the translational level. This translational regulation is implicated for many key regulators of cell fate and morphology in C. albicans. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  9. A 40 kd protein binds specifically to the 5'-untranslated regions of yeast mitochondrial mRNAs.

    PubMed Central

    Papadopoulou, B; Dekker, P; Blom, J; Grivell, L A

    1990-01-01

    Using a gel mobility shift assay we show that a 40 kd protein (p40), present in extracts of yeast mitochondria, binds specifically to the 5'-untranslated leader of cytochrome c oxidase subunit II mRNA. Binding of p40 to coxII RNA protects an 8-10 nucleotide segment from diethylpyocarbonate modification, indicating that the protein interacts with only a restricted region of the 5'-leader. This segment is located at position -12 with respect to the initiation AUG. Deletion of 10 nucleotides encompassing this site completely abolishes protein binding. Nevertheless, Bal31 deletion analysis within the coxII leader shows that a major part of the leader is essential for p40 binding, suggesting that binding of the protein is also dependent on secondary structural features. p40 binds to other mitochondrial leader mRNAs including those for coxI, coxIII and cyt b. p40 is present in a cytoplasmic (rho0) petite mutant lacking mitochondrial protein synthesis. It is therefore presumably nuclear encoded. The possible biological function of the protein is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. Fig. 9. PMID:1701144

  10. Stable and enhanced gene expression in Clostridium acetobutylicum using synthetic untranslated regions with a stem-loop.

    PubMed

    Lee, Joungmin; Jang, Yu-Sin; Papoutsakis, Eleftherios T; Lee, Sang Yup

    2016-07-20

    Gene overexpression is one of the most basic strategies in metabolic engineering, but the factors determining gene expression levels have been poorly studied in Clostridium species. In this study, we found that a short single-stranded 5' untranslated region (UTR) sequence led to decreased gene expression in Clostridium acetobutylicum. Using an in vitro enzyme assay and reverse transcription-quantitative PCR, we found that addition of a small stem-loop at the 5' end of mRNA increased mRNA levels and thereby protein expression levels up to 4.6-fold, possibly protecting mRNA from exonuclease attack. Gene-expression levels were apparently independent of the stability of the added stem-loop; the existence of a stem-loop itself appears to be more important. Our results indicate that efficient expression cassettes can be designed by taking the 5' UTR into consideration, as the expression levels can vary even though the same promoter and RBS are used. These findings will be useful for developing a more reliable gene expression system for metabolic engineering of Clostridium strains. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. CaMKIIalpha 3' untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage

    NASA Technical Reports Server (NTRS)

    Rook, M. S.; Lu, M.; Kosik, K. S.

    2000-01-01

    The CaMKIIalpha mRNA extends into distal hippocampal dendrites, and the 3' untranslated region (3'UTR) is sufficient to mediate this localization. We labeled the 3'UTR of the CaMKIIalpha mRNA in hippocampal cultures by using a green fluorescent protein (GFP)/MS2 bacteriophage tagging system. The CaMKIIalpha 3'UTR formed discrete granules throughout the dendrites of transfected cells. The identity of the fluorescent granules was verified by in situ hybridization. Over 30 min time periods these granules redistributed without a net increase in granule number; with depolarization there is a tendency toward increased numbers of granules in the dendrites. These observations suggest that finer time resolution of granule motility might reveal changes in the motility characteristics of granules after depolarization. So that motile granules could be tracked, shorter periods of observation were required. The movements of motile granules can be categorized as oscillatory, unidirectional anterograde, or unidirectional retrograde. Colocalization of CaMKIIalpha 3'UTR granules and synapses suggested that oscillatory movements allowed the granules to sample several local synapses. Neuronal depolarization increased the number of granules in the anterograde motile pool. Based on the time frame over which the granule number increased, the translocation of granules may serve to prepare the dendrite for mounting an adequate local translation response to future stimuli. Although the resident pool of granules can respond to signals that induce local translation, the number of granules in a dendrite might reflect its activation history.

  12. MicroRNAs and Androgen Receptor 3′ Untranslated Region: A Missing Link in Castration-resistant Prostate Cancer?

    PubMed Central

    Sikand, Kavleen; Barik, Sailen; Shukla, Girish C.

    2012-01-01

    The ligand-activated transcription factor, androgen receptor (AR) plays a central role in the development and progression of prostate cancer. Prostate cancer initiates as an androgen-dependent disease and further accumulation of multiple sequential genetic and epigenetic alterations transform it into an aggressive, castration-resistant prostate cancer (CRPC). The molecular basis of the transition from androgen-dependent prostate cancer to CRPC remains unclear. However, it is apparent that AR plays a pivotal role in this alteration. The recent discovery that microRNAs (miRNAs) can target the function of AR suggests a functional role of these non-coding RNAs in the pathogenesis of prostate cancer. miRNAs usually function by targeting the 3′ untranslated region (UTR) of a mRNA by base-pairing interactions and modulate translation either by destabilizing the message or by repression of protein synthesis in actively translating ribosomes. Here, we discuss the potential molecular pathways through which AR targeting miRNAs may promote CRPC. Modulation of AR expression by miRNAs presents a novel therapeutic option for prostate cancer, albeit it will likely be used in combination with the existing therapies. PMID:22468168

  13. Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

    PubMed Central

    Li, Zhaolong; Liu, Xin; Wang, Shaohua; Li, Jingliang; Hou, Min; Liu, Guanchen; Zhang, Wenyan; Yu, Xiao-Fang

    2016-01-01

    Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are two main causative pathogens of hand, foot and mouth disease (HFMD). Unlike EV71, virulence determinants of CA16, particularly within 5′ untranslated region (5′UTR), have not been investigated until now. Here, a series of nucleotides present in 5′UTR of lethal but not in non-lethal CA16 strains were screened by aligning nucleotide sequences of lethal circulating Changchun CA16 and the prototype G10 as well as non-lethal SHZH05 strains. A representative infectious clone based on a lethal Changchun024 sequence and infectious mutants with various nucleotide alterations in 5′UTR were constructed and further investigated by assessing virus replication in vitro and virulence in neonatal mice. Compared to the lethal infectious clone, the M2 mutant with a change from cytosine to uracil at nucleotide 104 showed weaker virulence and lower replication capacity. The predicted secondary structure of the 5′UTR of CA16 RNA showed that M2 mutant located between the cloverleaf and stem-loop II, affected interactions between the 5′UTR and the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and A1 (hnRNP A1) that are important for translational activity. Thus, our research determined a virulence-associated site in the 5′UTR of CA16, providing a crucial molecular target for antiviral drug development. PMID:26861413

  14. Long 5' untranslated regions regulate the RNA stability of the deep-sea filamentous phage SW1.

    PubMed

    Jian, Huahua; Xiong, Lei; Xu, Guanpeng; Xiao, Xiang; Wang, Fengping

    2016-02-22

    Virus production in the deep-sea environment has been found to be high, and viruses have been suggested to play significant roles in the overall functioning of this ecosystem. Nevertheless, little is known about these viruses, including the mechanisms that control their production, which makes them one of the least understood biological entities on Earth. Previously, we isolated the filamentous phage SW1, whose virus production and gene transcription were found to be active at low temperatures, from a deep-sea bacterium, Shewanella piezotolerans WP3. In this study, the operon structure of phage SW1 is presented, which shows two operons with exceptionally long 5' and 3' untranslated regions (UTRs). In addition, the 5'UTR was confirmed to significantly influence the RNA stability of the SW1 transcripts. Our study revealed novel regulation of the operon and led us to propose a unique regulatory mechanism for Inoviruses. This type of RNA-based regulation may represent a mechanism for significant viral production in the cold deep biosphere.

  15. Long 5′ untranslated regions regulate the RNA stability of the deep-sea filamentous phage SW1

    PubMed Central

    Jian, Huahua; Xiong, Lei; Xu, Guanpeng; Xiao, Xiang; Wang, Fengping

    2016-01-01

    Virus production in the deep-sea environment has been found to be high, and viruses have been suggested to play significant roles in the overall functioning of this ecosystem. Nevertheless, little is known about these viruses, including the mechanisms that control their production, which makes them one of the least understood biological entities on Earth. Previously, we isolated the filamentous phage SW1, whose virus production and gene transcription were found to be active at low temperatures, from a deep-sea bacterium, Shewanella piezotolerans WP3. In this study, the operon structure of phage SW1 is presented, which shows two operons with exceptionally long 5′ and 3′ untranslated regions (UTRs). In addition, the 5′UTR was confirmed to significantly influence the RNA stability of the SW1 transcripts. Our study revealed novel regulation of the operon and led us to propose a unique regulatory mechanism for Inoviruses. This type of RNA-based regulation may represent a mechanism for significant viral production in the cold deep biosphere. PMID:26898180

  16. Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli

    PubMed Central

    Cheol Kim, Seong; Eun Min, Byung; Gyu Hwang, Hyun; Woo Seo, Sang; Yeol Jung, Gyoo

    2015-01-01

    L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5′-untranslated region (5′-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5′-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria. PMID:26346938

  17. Modified ribosome profiling reveals high abundance of ribosome protected mRNA fragments derived from 3′ untranslated regions

    PubMed Central

    Miettinen, Teemu P.; Björklund, Mikael

    2015-01-01

    Ribosome profiling identifies ribosome positions on translated mRNAs. A prominent feature of published datasets is the near complete absence of ribosomes in 3′ untranslated regions (3′UTR) although substantial ribosome density can be observed on non-coding RNAs. Here we perform ribosome profiling in cultured Drosophila and human cells and show that different features of translation are revealed depending on the nuclease and the digestion conditions used. Most importantly, we observe high abundance of ribosome protected fragments in 3′UTRs of thousands of genes without manipulation of translation termination. Affinity purification of ribosomes indicates that the 3′UTR reads originate from ribosome protected fragments. Association of ribosomes with the 3′UTR may be due to ribosome migration through the stop codon or 3′UTR mRNA binding to ribosomes on the coding sequence. This association depends primarily on the relative length of the 3′UTR and may be related to translational regulation or ribosome recycling, for which the efficiency is known to inversely correlate with 3′UTR length. Together our results indicate that ribosome profiling is highly dependent on digestion conditions and that ribosomes commonly associate with the 3′UTR, which may have a role in translational regulation. PMID:25550424

  18. CaMKIIalpha 3' untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage

    NASA Technical Reports Server (NTRS)

    Rook, M. S.; Lu, M.; Kosik, K. S.

    2000-01-01

    The CaMKIIalpha mRNA extends into distal hippocampal dendrites, and the 3' untranslated region (3'UTR) is sufficient to mediate this localization. We labeled the 3'UTR of the CaMKIIalpha mRNA in hippocampal cultures by using a green fluorescent protein (GFP)/MS2 bacteriophage tagging system. The CaMKIIalpha 3'UTR formed discrete granules throughout the dendrites of transfected cells. The identity of the fluorescent granules was verified by in situ hybridization. Over 30 min time periods these granules redistributed without a net increase in granule number; with depolarization there is a tendency toward increased numbers of granules in the dendrites. These observations suggest that finer time resolution of granule motility might reveal changes in the motility characteristics of granules after depolarization. So that motile granules could be tracked, shorter periods of observation were required. The movements of motile granules can be categorized as oscillatory, unidirectional anterograde, or unidirectional retrograde. Colocalization of CaMKIIalpha 3'UTR granules and synapses suggested that oscillatory movements allowed the granules to sample several local synapses. Neuronal depolarization increased the number of granules in the anterograde motile pool. Based on the time frame over which the granule number increased, the translocation of granules may serve to prepare the dendrite for mounting an adequate local translation response to future stimuli. Although the resident pool of granules can respond to signals that induce local translation, the number of granules in a dendrite might reflect its activation history.

  19. Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon.

    PubMed

    Karlsen, Marius; Villoing, Stephane; Rimstad, Espen; Nylund, Are

    2009-10-27

    Salmonid alphavirus (SAV) causes disease in farmed salmonid fish and is divided into different genetic subtypes (SAV1-6). Here we report the cloning and characterization of the 5'- and 3'- untranslated regions (UTR) of a SAV3 isolated from Atlantic salmon in Norway. The sequences of the UTRs are very similar to those of SAV1 and SAV2, but single nucleotide polymorphisms are present, also in the 3' - conserved sequence element (3'-CSE). Prediction of the RNA secondary structure suggested putative stem-loop structures in both the 5'- and 3'-ends, similar to those of alphaviruses from the terrestrial environment, indicating that the general genome replication initiation strategy for alphaviruses is also utilized by SAV. A DNA replicon vector, pmSAV3, based upon a pVAX1 backbone and the SAV3 genome was constructed, and the SAV3 non-structural proteins were used to express a reporter gene controlled by the SAV3 subgenomic promoter. Transfection of pmSAV3 into CHSE and BF2 cell lines resulted in expression of the reporter protein, confirming that the cloned SAV3 replication apparatus and UTRs are functional in fish cells.

  20. The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

    PubMed Central

    Moncini, Silvia; Bevilacqua, Annamaria; Venturin, Marco; Fallini, Claudia; Ratti, Antonia; Nicolin, Angelo; Riva, Paola

    2007-01-01

    Background CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression. Results The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins. Conclusion Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders. PMID:18053171

  1. Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

    PubMed

    Bermejo, B; Remacha, M; Ortiz-Reyes, B; Santos, C; Ballesta, J P

    1994-02-11

    Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

  2. 5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

    PubMed Central

    Diamos, Andrew G.; Rosenthal, Sun H.; Mason, Hugh S.

    2016-01-01

    We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody

  3. The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus modulates infection.

    PubMed

    Kerr, Craig H; Wang, Qing S; Keatings, Kathleen; Khong, Anthony; Allan, Douglas; Yip, Calvin K; Foster, Leonard J; Jan, Eric

    2015-06-01

    Dicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5' untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells. Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in

  4. Reselection of a genomic upstream open reading frame in mouse hepatitis coronavirus 5'-untranslated-region mutants.

    PubMed

    Wu, Hung-Yi; Guan, Bo-Jhih; Su, Yu-Pin; Fan, Yi-Hsin; Brian, David A

    2014-01-01

    An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5' untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5'-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5'-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.

  5. Far Upstream Element-Binding Protein 1 Binds the 3' Untranslated Region of PKD2 and Suppresses Its Translation.

    PubMed

    Zheng, Wang; Shen, Fan; Hu, Ruikun; Roy, Birbickram; Yang, JungWoo; Wang, Qian; Zhang, Fan; King, Jennifer C; Sergi, Consolato; Liu, Song-Mei; Cordat, Emmanuelle; Tang, Jingfeng; Cao, Ying; Ali, Declan; Chen, Xing-Zhen

    2016-09-01

    Autosomal dominant polycystic kidney disease pathogenesis can be recapitulated in animal models by gene mutations in or dosage alterations of polycystic kidney disease 1 (PKD1) or PKD2, demonstrating that too much and too little PKD1/PKD2 are both pathogenic. Gene dosage manipulation has become an appealing approach by which to compensate for loss or gain of gene function, but the mechanisms controlling PKD2 expression remain incompletely characterized. In this study, using cultured mammalian cells and dual-luciferase assays, we found that the 3' untranslated region (3'UTR) of PKD2 mRNA inhibits luciferase protein expression. We then identified nucleotides 691-1044, which we called 3FI, as the 3'UTR fragment necessary for repressing the expression of luciferase or PKD2 in this system. Using a pull-down assay and mass spectrometry we identified far upstream element-binding protein 1 (FUBP1) as a 3FI-binding protein. In vitro overexpression of FUBP1 inhibited the expression of PKD2 protein but not mRNA. In embryonic zebrafish, FUBP1 knockdown (KD) by morpholino injection increased PKD2 expression and alleviated fish tail curling caused by morpholino-mediated KD of PKD2. Conversely, FUBP1 overexpression by mRNA injection significantly increased pronephric cyst occurrence and tail curling in zebrafish embryos. Furthermore, FUBP1 binds directly to eukaryotic translation initiation factor 4E-binding protein 1, indicating a link to the translation initiation complex. These results show that FUBP1 binds 3FI in the PKD2 3'UTR to inhibit PKD2 translation, regulating zebrafish disease phenotypes associated with PKD2 KD. Copyright © 2016 by the American Society of Nephrology.

  6. Association of a duplicated repeat polymorphism in the 5'-untranslated region of the DRD4 gene with novelty seeking.

    PubMed

    Rogers, Geraldine; Joyce, Peter; Mulder, Roger; Sellman, Douglas; Miller, Allison; Allington, Melanie; Olds, Robin; Wells, Elisabeth; Kennedy, Martin

    2004-04-01

    Novelty Seeking (NS) is a human personality trait in which impulsive, exploratory, and thrill-seeking behaviors are displayed. Dopaminergic genes have been prime candidates in the search for the genetic factors underlying NS because of the central role that dopamine plays in the brain's reward system. We have investigated whether there is an association between a polymorphic 120 base pairs (bp) repeat that is located in the 5'-untranslated region of the dopamine D4 receptor gene (DRD4) and NS. We genotyped four separate groups from psychiatric clinical studies for the repeat polymorphism. There were significant associations with NS in the groups of bipolar (P = 0.01) and alcoholic (P = 0.006) families containing 267 and 172 subjects, respectively. Subjects who were homozygous for the single-copy allele (SS genotype) had higher mean NS scores. This trend was also observed in the two other studies that contained unrelated subjects diagnosed with depression (N = 143 and N = 148) but the associations between DRD4 duplication genotype and NS were not significant in these groups. In the data combined from all four clinical groups those genotyped as SS had higher mean scores for all four NS subscales with significant associations for impulsivity (P = 0.0006), extravagance (P = 0.04), disorderliness (P = 0.02), and total NS (P = 0.0003). However, given the low frequency of the single-copy allele, this polymorphism would account for only a small proportion of the variance of NS in the population. Copyright 2003 Wiley-Liss, Inc.

  7. PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

    PubMed Central

    Espinosa-Hernández, Wendy; Velez-Uriza, Dora; Valdés, Jesús; Vélez-Del Valle, Cristina; Salas-Benito, Juan; Martínez-Contreras, Rebeca; García-Espítia, Matilde; Salas-Benito, Mariana; Vega-Almeida, Tania; De Nova-Ocampo, Mónica

    2014-01-01

    The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication. PMID:25406089

  8. Destabilization of ERBB2 transcripts by targeting 3' untranslated region messenger RNA associated HuR and histone deacetylase-6.

    PubMed

    Scott, Gary K; Marx, Corina; Berger, Crystal E; Saunders, Laura R; Verdin, Eric; Schäfer, Stefan; Jung, Manfred; Benz, Christopher C

    2008-07-01

    In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies.

  9. Chikungunya Virus 3′ Untranslated Region: Adaptation to Mosquitoes and a Population Bottleneck as Major Evolutionary Forces

    PubMed Central

    Chen, Rubing; Wang, Eryu; Tsetsarkin, Konstantin A.; Weaver, Scott C.

    2013-01-01

    The 3′ untranslated genome region (UTR) of arthropod-borne viruses is characterized by enriched direct repeats (DRs) and stem-loop structures. Despite many years of theoretical and experimental study, on-going positive selection on the 3′UTR had never been observed in ‘real-time,’ and the role of the arbovirus 3′UTR remains poorly understood. We observed a lineage-specific 3′UTR sequence pattern in all available Asian lineage of the mosquito-borne alphavirus, chikungunya virus (CHIKV) (1958–2009), including complicated mutation and duplication patterns of the long DRs. Given that a longer genome is usually associated with less efficient replication, we hypothesized that the fixation of these genetic changes in the Asian lineage 3′UTR was due to their beneficial effects on adaptation to vectors or hosts. Using reverse genetic methods, we examined the functional importance of each direct repeat. Our results suggest that adaptation to mosquitoes, rather than to mammalian hosts, is a major evolutionary force on the CHIKV 3′UTR. Surprisingly, the Asian 3′UTR appeared to be inferior to its predicted ancestral sequence for replication in both mammals and mosquitoes, suggesting that its fixation in Asia was not a result of directional selection. Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia. We propose that this introduction of a 3′UTR with deletions led to genetic drift and compensatory mutations associated with the loss of structural/functional constraints, followed by two independent beneficial duplications and fixation due to positive selection. Our results provide further evidence that the limited epidemic potential of the Asian CHIKV strains resulted from founder effects that reduced its fitness for efficient transmission by mosquitoes there. PMID:24009512

  10. A eukaryotic-like 3′ untranslated region in Salmonella enterica hilD mRNA

    PubMed Central

    López-Garrido, Javier; Puerta-Fernández, Elena; Casadesús, Josep

    2014-01-01

    Long 3′ untranslated regions (3′UTRs) are common in eukaryotic mRNAs. In contrast, long 3′UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3′UTR of 310 nucleotides in hilD mRNA, a transcript that encodes a transcriptional activator of Salmonella enterica pathogenicity island 1 (SPI-1). Deletion of the hilD 3′UTR increases the hilD mRNA level, suggesting that the hilD 3′UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3′UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3′UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3′UTR mRNAs, suggesting that the hilD 3′UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3′UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3′UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3′UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover. PMID:24682814

  11. PTB binds to the 3' untranslated region of the human astrovirus type 8: a possible role in viral replication.

    PubMed

    Espinosa-Hernández, Wendy; Velez-Uriza, Dora; Valdés, Jesús; Vélez-Del Valle, Cristina; Salas-Benito, Juan; Martínez-Contreras, Rebeca; García-Espítia, Matilde; Salas-Benito, Mariana; Vega-Almeida, Tania; De Nova-Ocampo, Mónica

    2014-01-01

    The 3' untranslated region (3'UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3'UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3'UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3'UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3'UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3'UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3'UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3'UTR HAstV-8 and is required or participates in viral replication.

  12. The Modulatory Effects of the Polymorphisms in GLA 5'-Untranslated Region Upon Gene Expression Are Cell-Type Specific.

    PubMed

    Ferreira, Susana; Reguenga, Carlos; Oliveira, João Paulo

    2015-01-01

    Lysosomal α-galactosidase A (αGal) is the enzyme deficient in Fabry disease (FD). The 5'-untranslated region (5'UTR) of the αGal gene (GLA) shows a remarkable degree of variation with three common single nucleotide polymorphisms at nucleotide positions c.-30G>A, c.-12G>A and c.-10C>T. We have recently identified in young Portuguese stroke patients a fourth polymorphism, at c.-44C>T, co-segregating in cis with the c.-12A allele. In vivo, the c.-30A allele is associated with higher enzyme activity in plasma, whereas c.-10T is associated with moderately decreased enzyme activity in leucocytes. Limited data suggest that c.-44T might be associated with increased plasma αGal activity. We have used a luciferase reporter system to experimentally assess the relative modulatory effects on gene expression of the different GLA 5'UTR polymorphisms, as compared to the wild-type sequence, in four different human cell lines. Group-wise, the relative luciferase expression patterns of the various GLA variant isoforms differed significantly in all four cell lines, as evaluated by non-parametric statistics, and were cell-type specific. Some of the post hoc pairwise statistical comparisons were also significant, but the observed effects of the GLA 5'UTR polymorphisms upon the luciferase transcriptional activity in vitro did not consistently replicate the in vivo observations.These data suggest that the GLA 5'UTR polymorphisms are possible modulators of the αGal expression. Further studies are needed to elucidate the biological and clinical implications of these observations, particularly to clarify the effect of these polymorphisms in individuals carrying GLA variants associated with high residual enzyme activity, with no or mild FD clinical phenotypes.

  13. Regulatory Feedback Loop of Two phz Gene Clusters through 5′-Untranslated Regions in Pseudomonas sp. M18

    PubMed Central

    Li, Yaqian; Du, Xilin; Lu, Zhi John; Wu, Daqiang; Zhao, Yilei; Ren, Bin; Huang, Jiaofang; Huang, Xianqing; Xu, Yuhong; Xu, Yuquan

    2011-01-01

    Background Phenazines are important compounds produced by pseudomonads and other bacteria. Two phz gene clusters called phzA1-G1 and phzA2-G2, respectively, were found in the genome of Pseudomonas sp. M18, an effective biocontrol agent, which is highly homologous to the opportunistic human pathogen P. aeruginosa PAO1, however little is known about the correlation between the expressions of two phz gene clusters. Methodology/Principal Findings Two chromosomal insertion inactivated mutants for the two gene clusters were constructed respectively and the correlation between the expressions of two phz gene clusters was investigated in strain M18. Phenazine-1-carboxylic acid (PCA) molecules produced from phzA2-G2 gene cluster are able to auto-regulate expression itself and activate the expression of phzA1-G1 gene cluster in a circulated amplification pattern. However, the post-transcriptional expression of phzA1-G1 transcript was blocked principally through 5′-untranslated region (UTR). In contrast, the phzA2-G2 gene cluster was transcribed to a lesser extent and translated efficiently and was negatively regulated by the GacA signal transduction pathway, mainly at a post-transcriptional level. Conclusions/Significance A single molecule, PCA, produced in different quantities by the two phz gene clusters acted as the functional mediator and the two phz gene clusters developed a specific regulatory mechanism which acts through 5′-UTR to transfer a single, but complex bacterial signaling event in Pseudomonas sp. strain M18. PMID:21559370

  14. A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA.

    PubMed Central

    Hellendoorn, K; Verlaan, P W; Pleij, C W

    1997-01-01

    The 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5' UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops. Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants. Different symptoms were observed for the various mutants, pointing to a functional role of the C-C and C-A mismatches in the hairpins of the 5' UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants. Remarkably, in all cases C-C or C-A mismatches were introduced. The internal loop of the 5'-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin. A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed. PMID:9343237

  15. Fragile X mental retardation protein interactions with a G quadruplex structure in the 3'-untranslated region of NR2B mRNA.

    PubMed

    Stefanovic, Snezana; DeMarco, Brett A; Underwood, Ayana; Williams, Kathryn R; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-12-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5'-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the post synaptic density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3'-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3'-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-d-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets.

  16. Porcine SOX9 Gene Expression Is Influenced by an 18 bp Indel in the 5'-Untranslated Region.

    PubMed

    Brenig, Bertram; Duan, Yanyu; Xing, Yuyun; Ding, Nengshui; Huang, Lusheng; Schütz, Ekkehard

    2015-01-01

    Sex determining region Y-box 9 (SOX9) is an important regulator of sex and skeletal development and is expressed in a variety of embryonal and adult tissues. Loss or gain of function resulting from mutations within the coding region or chromosomal aberrations of the SOX9 locus lead to a plethora of detrimental phenotypes in humans and animals. One of these phenotypes is the so-called male-to-female or female-to-male sex-reversal which has been observed in several mammals including pig, dog, cat, goat, horse, and deer. In 38,XX sex-reversal French Large White pigs, a genome-wide association study suggested SOX9 as the causal gene, although no functional mutations were identified in affected animals. However, besides others an 18 bp indel had been detected in the 5'-untranslated region of the SOX9 gene by comparing affected animals and controls. We have identified the same indel (Δ18) between position +247 bp and +266 bp downstream the transcription start site of the porcine SOX9 gene in four other pig breeds; i.e., German Large White, Laiwu Black, Bamei, and Erhualian. These animals have been genotyped in an attempt to identify candidate genes for porcine inguinal and/or scrotal hernia. Because the 18 bp segment in the wild type 5'-UTR harbours a highly conserved cAMP-response element (CRE) half-site, we analysed its role in SOX9 expression in vitro. Competition and immunodepletion electromobility shift assays demonstrate that the CRE half-site is specifically recognized by CREB. Both binding of CREB to the wild type as well as the absence of the CRE half-site in Δ18 reduced expression efficiency in HEK293T, PK-15, and ATDC5 cells significantly. Transfection experiments of wild type and Δ18 SOX9 promoter luciferase constructs show a significant reduction of RNA and protein levels depending on the presence or absence of the 18 bp segment. Hence, the data presented here demonstrate that the 18 bp indel in the porcine SOX9 5'-UTR is of functional importance and may

  17. The 5′ untranslated regions (UTRs) of CCN1, CCN2, and CCN4 exhibit cryptic promoter activity

    PubMed Central

    Huang, Bau-Lin; Dornbach, Lisa M.

    2007-01-01

    CCNs are structurally related matricellular proteins that are highly expressed in many embryonic and adult tissues, including the skeletal system and tumors, where canonical cap-dependent translation is suppressed under hypoxic environments. CCNs are encoded by mRNAs containing long G/C rich 5′-untranslated regions (5′-UTRs). Given that they are expressed under conditions of cellular stress, it has been suggested that the long G/C-rich regions contain internal ribosomal entry sites (IRES) that allow these mRNAS to be translated under conditions where cap-dependent translation is suppressed. Previously published work supported this possibility. However, recent studies have shown that a number of previously reported cellular IRES elements do not in fact possess IRES activity. Here we aimed to reveal whether the 5′UTRs of CCNs harbor IRES activities. The 5′UTRs of CCN1, 2, and 4 were tested in this study. Our results showed that the 5′UTRs of these genes do not contain IRES elements, but instead appear to contain cryptic promoters. Both promoterless and hairpin-containing dicistronic tests showed that transcription was initiated by cryptic promoter elements in 5′UTRs of CCN1, 2, and 4. When dicistronic mRNAs were translated in vitro or in vivo, no IRES activities were detected in the 5′UTRs of CCN1, 2, and 4. Furthermore, these cryptic promoter activities from 5′UTRs of CCN1, 2, and 4 could be detected in various cell types, including chondrocytes, osteoblasts, and endothelial cells, where the cryptic promoter permitted varying degrees of activation. In addition, the core promoter element of the CCN2 5′UTR was identified. CCNs are expressed under conditions of cellular stress, and it has been suggested that some CCN family members utilize IRES-mediated translation initiation to facilitate this expression. We found no evidence for IRES activity, but rather found that the unusually long 5′UTRs of CCNs 1, 2, and 4 harbor cryptic promoters that showed

  18. In silico analysis of microRNAS targeting the HLA-G 3' untranslated region alleles and haplotypes.

    PubMed

    Castelli, Erick C; Moreau, Philippe; Oya e Chiromatzo, Alynne; Mendes-Junior, Celso Teixeira; Veiga-Castelli, Luciana C; Yaghi, Layale; Giuliatti, Silvana; Carosella, Edgardo D; Donadi, Eduardo Antonio

    2009-12-01

    It has been reported that microRNAs (miRNA) may have allele-specific targeting for the 3' untranslated region (3' UTR) of the HLA-G locus. In a previous study, we reported 11 3'UTR haplotypes encompassing the 14-bp insertion/deletion polymorphism and seven SNPs (+3003 T/C, +3010 C/G, +3027 C/A, +3035 C/T, +3142 C/G, +3187 A/G, and +3196 C/G), of which only the +3142 C/G SNP has been reported to influence the binding of miRNAs. Using bioinformatics analyses, we identified putative miRNA-binding sites considering the haplotypes encompassing these eight polymorphic sites, and we ranked the lowest free energies that could potentially lead to an mRNA degradation or translational repression. When a specific haplotype or a particular SNP was associated with a miRNA-binding site, we defined a free energy difference of 4 kcal/mol between alleles to classify them energetically distant. The best results were obtained for the miR-513a-5p, miR-518c*, miR-1262 and miR-92a-1*, miR-92a-2*, miR-661, miR-1224-5p, and miR-433 miRNAs, all influencing one or more of the +3003, +3010, +3027, and +3035 SNPs. The miR-2110, miR-93, miR-508-5p, miR-331-5p, miR-616, miR-513b, and miR-589* miRNAs targeted the 14-bp fragment region, and miR-148a, miR-19a*, miR-152, mir-148b, and miR-218-2 also influenced the +3142 C/G polymorphism. These results suggest that these miRNAs might play a relevant role on the HLA-G expression pattern.

  19. Partial deletion of stem-loop 2 in the 3' untranslated region of foot-and-mouth disease virus identifies a region that is dispensable for virus replication.

    PubMed

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    The 3' untranslated region (3' UTR) of the foot-and-mouth disease virus (FMDV) genome plays an essential role in virus replication, but the properties of the 3' UTR are not completely defined. In order to determine the role of different regions of the 3' UTR in FMDV replication, we conducted site-directed mutagenesis of the 3' UTR of FMDV serotype O IND R2/1975 using a cDNA clone. Through independent serial deletions in various regions of the 3' UTR, we demonstrated that deletion of nucleotides between the stem-loop (SL) structures and in the beginning and the end regions of the SL2 structure could be lethal for FMDV replication. However, a block deletion of 20 nucleotides (nt 60 to 79) in the middle of SL2 did not affect the viability of FMDV in cultured cells. Characterisation of the deletion mutant virus (O(R2/1975-Δ3'UTR 60-79)) revealed no significant difference in growth kinetics or RNA replication ability compared to the parental virus. However, the mutant virus produced slightly larger plaques when compared to the parental virus. This is the first description of a dispensable 20-nucleotide region in SL2 of the FMDV 3' UTR.

  20. Functional Characterization of a Single Nucleotide Polymorphism in the 3' Untranslated Region of Sheep DLX3 Gene.

    PubMed

    Rong, Enguang; Zhang, Zhiwei; Qiao, Shupei; Yang, Hua; Yan, Xiaohong; Li, Hui; Wang, Ning

    2015-01-01

    The Distal-less 3 (homeobox protein DLX-3), a transcription factor, is critical for the development of hair follicle and hair formation and regeneration. We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3' untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown. In the present study, we performed association analysis between these four identified SNPs and DLX3 gene expression in sheep skin using quantitative real-time RT-PCR. The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels. Then, we constructed DLX3 3'UTR luciferase reporters and validated the association. The reporter assays showed that the three major haplotypes, derived from the four SNPs, had significantly different effects on luciferase reporter activity and the four SNPs also had significantly different allelic effects on the luciferase reporter activity (p < 0.05). Bioinformatics analysis showed that the SNP (c. *1,038_1,039 insC) was located within a potential miR-188 binding site of the 3'UTR of sheep DLX3 mRNA. This SNP may affect miR-188-mediated DLX3 gene expression and result in phenotypic variation. To test the hypothesis, we investigated the effects of miR-188 mimic and inhibitor on the activity of the DLX3 3'UTR luciferase reporter with different SNP alleles. The results showed that in both sheep fetal fibroblasts (SFFs) and human HaCaT cells, miR-188 mimic could significantly decrease the allele D (deletion) luciferase reporter activity (p < 0.05), but miR-188 inhibitor could increased the reporter activitiy. However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity. In addition, transfection of miR-188 mimic dramatically decreased the endogenous

  1. Functional Characterization of a Single Nucleotide Polymorphism in the 3' Untranslated Region of Sheep DLX3 Gene

    PubMed Central

    Rong, Enguang; Zhang, Zhiwei; Qiao, Shupei; Yang, Hua; Yan, Xiaohong; Li, Hui; Wang, Ning

    2015-01-01

    The Distal-less 3 (homeobox protein DLX-3), a transcription factor, is critical for the development of hair follicle and hair formation and regeneration. We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3′ untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown. In the present study, we performed association analysis between these four identified SNPs and DLX3 gene expression in sheep skin using quantitative real-time RT-PCR. The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels. Then, we constructed DLX3 3′UTR luciferase reporters and validated the association. The reporter assays showed that the three major haplotypes, derived from the four SNPs, had significantly different effects on luciferase reporter activity and the four SNPs also had significantly different allelic effects on the luciferase reporter activity (p < 0.05). Bioinformatics analysis showed that the SNP (c. *1,038_1,039 insC) was located within a potential miR-188 binding site of the 3′UTR of sheep DLX3 mRNA. This SNP may affect miR-188-mediated DLX3 gene expression and result in phenotypic variation. To test the hypothesis, we investigated the effects of miR-188 mimic and inhibitor on the activity of the DLX3 3′UTR luciferase reporter with different SNP alleles. The results showed that in both sheep fetal fibroblasts (SFFs) and human HaCaT cells, miR-188 mimic could significantly decrease the allele D (deletion) luciferase reporter activity (p < 0.05), but miR-188 inhibitor could increased the reporter activitiy. However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity. In addition, transfection of miR-188 mimic dramatically decreased the

  2. MUTATIONS IN THE E2 GLYCOPROTEIN AND THE 3' UNTRANSLATED REGION ENHANCE CHIKUNGUNYA VIRUS VIRULENCE IN MICE.

    PubMed

    Hawman, David W; Carpentier, Kathryn S; Fox, Julie M; May, Nicholas A; Sanders, Wes; Montgomery, Stephanie A; Moorman, Nathaniel J; Diamond, Michael S; Morrison, Thomas E

    2017-07-26

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes debilitating musculoskeletal pain and inflammation and can persist for months to years after acute infection. Although studies in humans and experimentally-infected animals suggest that CHIKV infection persists in musculoskeletal tissues, the mechanisms for this remain poorly understood. To evaluate this further, we isolated CHIKV from the serum of persistently infected Rag1(-/-) mice at day 28. When inoculated into naïve WT mice, this persistently circulating CHIKV strain displayed a capacity for earlier dissemination and greater pathogenicity compared with the parental virus. Sequence analysis revealed a nonsynonymous mutation in the E2 glycoprotein (E2 K200R) and a deletion within the 3' untranslated region (3' -UTR). Introduction of these changes into the parental virus conferred enhanced virulence in mice although a primary tropism for musculoskeletal tissues was maintained. The E2 K200R mutation was largely responsible for enhanced viral dissemination and pathogenicity, although these effects were augmented by the 3' -UTR deletion. Finally, studies in Irf3/Irf7(-/-) and Ifnar1(-/-) mice suggest that the E2 K200R mutation enhances viral dissemination from the site of inoculation independently of IRF3, IRF7, and IFNAR1 mediated responses. As our findings reveal viral determinants of CHIKV dissemination and pathogenicity, their further study should help to elucidate host-virus interactions that determine acute and chronic CHIKV infection.IMPORTANCE CHIKV is a globally-spreading, mosquito-transmitted virus that causes debilitating acute and chronic musculoskeletal disease in humans. The viral genetic determinants that dictate acute and chronic disease severity are not understood. To improve our understanding of CHIKV pathogenesis, we evaluated a CHIKV strain isolated from the serum of chronically-infected immunocompromised mice. Sequence analysis of this persistent CHIKV strain identified

  3. The parvovirus H-1 NS2 protein affects viral gene expression through sequences in the 3' untranslated region.

    PubMed

    Li, X; Rhode, S L

    1993-05-01

    We reported previously that an NS2 null mutant of parvovirus H-1 (H-1SA) was capable of lytic growth in human and hamster cells, but not in rat cells (Li and Rhode, 1991). The host-range phenotype of H-1SA was also manifested in newborn rats and was associated with a reduction of viral protein synthesis to about 10% of wild-type virus and an absence of virions in cultured rat fibroblasts. However, the H-1SA mRNAs for NS1 and capsid proteins, R1 and R3, accumulated to wild-type levels and translated well with a cell free rabbit reticulocyte lysate. These results indicate that NS2 plays an important role in the regulation of viral protein synthesis in rat cells in vivo and in vitro, but NS2 is largely dispensable in other types of cells, such as human and hamster cells. To analyze whether the 5' and 3' untranslated regions (UTR) of viral RNA are involved in the regulation by NS2, the viral VP2 gene was replaced by a reporter gene, firefly luciferase, in a plasmid clone of viral sequences and the protein synthesis under the control of P38 was evaluated by luciferase assay. Cells were transfected with luciferase expressing plasmids and subsequently infected with wild-type H-1 or H-1SA. We were able to mimic the defect in expression that we observed in cultured cells and animals with virus infection. Luciferase activity in H- 1SA-infected rat cells was about 10-fold lower than that in H-1-infected rat cells, but only 2-fold lower or less in H-1SA-infected human cells and hamster cells compared to wild-type H-1. These results are consistent with our previous data that NS2 has a host-range phenotype in the natural host of H-1, the rat. Deletion of 5' UTR sequences from P38 transcripts reduced the overall P38-luc expression but expression was NS2 independent, whereas deletion of the terminal 3' UTR sequences of viral RNA reduced NS2-dependent expression in rat cells. These results suggest that the regulation of viral protein synthesis by NS2 depends on RNA sequences in the

  4. The 5' untranslated region and Gag product of Idefix, a long terminal repeat-retrotransposon from Drosophila melanogaster, act together to initiate a switch between translated and untranslated states of the genomic mRNA.

    PubMed

    Meignin, Carine; Bailly, Jean-Luc; Arnaud, Frédérick; Dastugue, Bernard; Vaury, Chantal

    2003-11-01

    Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5' untranslated region (5'UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5'UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5'UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.

  5. Polymorphisms in the 3'-untranslated region of the CDH1 gene are a risk factor for primary gastric diffuse large B-cell lymphoma.

    PubMed

    Jacobs, Gunnar; Hellmig, Stephan; Huse, Klaus; Titz, Andrea; Franke, Andre; Kwiatkowski, Ruta; Ott, Stephan; Kosmahl, Markus; Fischbach, Wolfgang; Lucius, Ralph; Klapper, Wolfram; Fölsch, Ulrich R; Hampe, Jochen; Schreiber, Stefan; Rosenstiel, Philip

    2011-07-01

    Primary gastric B-cell lymphomas arise from mucosa-associated lymphatic tissue (MALT) in patients with chronic Helicobacter pylori infection. We investigated whether germline variants in the CDH1 gene, coding for E-cadherin, genetically predispose patients to primary gastric B-cell lymphoma. Single marker analyses of the CDH1 gene were conducted in patients with primary gastric B-cell lymphoma (n=144), in patients with primary gastric high-grade lymphoma (n=61), and in healthy blood donors (n=361). Twelve single nucleotide polymorphisms were genotyped by TaqMan(®) technology. Allelic imbalance was tested by pyrosequencing and clone direct sequencing of heterozygote genomic and cDNA. Mutation detection was conducted around the poly-A signal of the CDH1 3'-untranslated region. The influence of the 3'-untranslated region on protein translation was determined by a luciferase reporter assay. Single marker analyses identified two single nucleotide polymorphisms in strong linkage disequilibrium located in the CDH1 3'-untranslated region. One of them was significantly associated with primary gastric diffuse large B-cell lymphomas after correction for multiple testing and this association was confirmed in an independent sample set. Patients homozygous for the rare T allele (rs1801026) had a 4.9-fold increased risk (95% CI: 1.5-15.9) of developing primary gastric diffuse large B-cell lymphoma. Allelic imbalance and reporter gene assays indicated a putative influence on mRNA stability and/or translational efficacy. We identified variants in CDH1 as the first potential genetic risk factors for the development of primary gastric diffuse large B-cell lymphomas. One of the potentially causative variants affects allelic CDH1 expression. These findings support the hypothesis that besides somatic alterations of B-cells, germline variants in the CDH1 gene contribute to a predisposition to the development of primary gastric diffuse large B-cell lymphomas.

  6. Highly accessible AU-rich regions in 3’ untranslated regions are hotspots for binding of regulatory factors

    PubMed Central

    2017-01-01

    Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3’UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing “free” target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer

  7. Is single-strand conformation polymorphism analysis of the full 5' untranslated region an adequate approach to study hepatitis C virus quasispecies distribution?

    PubMed

    Vera-Otarola, Jorge; Barría, María Inés; León, Ursula; Carvallo, Pilar; Soza, Alejandro; López-Lastra, Marcelo

    2009-09-01

    Single-strand conformation polymorphism (SSCP) analysis is used by many laboratories to study the quasispecies distribution of the hepatitis C virus (HCV). Here we question the validity of this experimental approach, as conclusions are drawn from the analysis of the migration patterns of two ssDNA molecules and not from RNA. Using previously characterized mutants of the HCV 5' untranslated regions, we show that contrary to what has been predicted, SSCP migration patterns of DNA amplicons with differences in their nucleotide sequences generated from the full 5' UTR of HCV are not necessarily unique.

  8. Is Single-Strand Conformation Polymorphism Analysis of the Full 5′ Untranslated Region an Adequate Approach To Study Hepatitis C Virus Quasispecies Distribution? ▿

    PubMed Central

    Vera-Otarola, Jorge; Barría, María Inés; León, Ursula; Carvallo, Pilar; Soza, Alejandro; López-Lastra, Marcelo

    2009-01-01

    Single-strand conformation polymorphism (SSCP) analysis is used by many laboratories to study the quasispecies distribution of the hepatitis C virus (HCV). Here we question the validity of this experimental approach, as conclusions are drawn from the analysis of the migration patterns of two ssDNA molecules and not from RNA. Using previously characterized mutants of the HCV 5′ untranslated regions, we show that contrary to what has been predicted, SSCP migration patterns of DNA amplicons with differences in their nucleotide sequences generated from the full 5′ UTR of HCV are not necessarily unique. PMID:19553315

  9. Analysis of the H gene, the central untranslated region and the proximal coding part of the F gene of wild-type and vaccine canine distemper viruses.

    PubMed

    Haas, L; Liermann, H; Harder, T C; Barrett, T; Löchelt, M; von Messling, V; Baumgärtner, W; Greiser-Wilke, I

    1999-09-01

    This paper summarizes the results of the genetic analysis of several parts of the genome of canine distemper virus (CDV) field isolates and vaccine viruses. The haemagglutinin (H) gene analysis showed that recent viruses did not differ significantly from vaccine strains. The analysis of the long untranslated region between the matrix (M) and fusion (F) gene revealed distinct genetic heterogeneity. The putative F protein start codon AUG461 of vaccine strain Onderstepoort was found to be mutated in all wild-type isolates and in another vaccine strain. The proximal coding part of the F gene was well conserved. Phylogenetic analysis of this segment showed the presence of several cocirculating CDV genotypes.

  10. Structure and expression of the guinea pig preproenkephalin gene: site-specific cleavage in the 3' untranslated region yields truncated mRNA transcripts in specific brain regions.

    PubMed Central

    LaForge, K S; Unterwald, E M; Kreek, M J

    1995-01-01

    We isolated the guinea pig preproenkephalin gene from a genomic library by hybridization to a rat cDNA probe. The entire nucleotide sequence of the gene was determined. Genomic Southern blot hybridization demonstrated that the gene exists in a single copy within the genome. On the basis of RNase protection transcript mapping and homology comparisons with known preproenkephalin sequences from other species and assuming a poly(A) tail length of 100 residues, we predicted an mRNA transcript of approximately 1,400 nucleotides encoded by three exons. Northern (RNA) blot analysis of total RNA from several brain regions showed high levels of preproenkephalin mRNA in the caudate putamen, nucleus accumbens, and hypothalamus, with detectable levels in the amygdala, ventral tegmental area, and central gray and also in the pituitary. Unexpectedly, in several brain regions, the mRNA appeared not only in the 1,400-nucleotide length but also in a shorter length of approximately 1,130 bases. Significant amounts of the shorter mRNA were found in the caudate putamen, nucleus accumbens, and amygdala. The longer, but not the shorter, transcripts from the caudate putamen were found to be polyadenylated, but the difference in size was not due solely to the presence of poly(A) tails. Northern gel analysis of total RNA from the caudate putamen with probes from each exon, together with RNase protection mapping of the 3' end of the mRNA demonstrated that the 1,400-base preproenkephalin mRNA transcripts are cleaved in a site-specific manner in some brain regions, yielding a 1,130-base transcript and a 165-base polyadenylated fragment derived from the terminal end of the 3' untranslated region of the mRNA. This cleavage may serve as a preliminary step in RNA degradation and provide a mechanism for control of preproenkephalin mRNA abundance through selective degradation. PMID:7891703

  11. The 5' untranslated region of the VR-ACS1 mRNA acts as a strong translational enhancer in plants.

    PubMed

    Wever, Willem; McCallum, Emily J; Chakravorty, David; Cazzonelli, Christopher I; Botella, José R

    2010-08-01

    The structure and function of untranslated mRNA leader sequences and their role in controlling gene expression remains poorly understood. Previous research has suggested that the 5' untranslated region (5'UTR) of the Vigna radiata aminocyclopropane-1-carboxylate synthase synthase (VR-ACS1) gene may function as a translational enhancer in plants. To test such hypothesis we compared the translation enhancing properties of three different 5'UTRs; those from the VR-ACS1, the chlorophyll a/b binding gene from petunia (Cab22L; a known translational enhancer) and the Vigna radiata pectinacetylesterase gene (PAE; used as control). Identical constructs in which the coding region of the beta-glucuronidase (GUS) gene was fused to each of the three 5'UTRs and placed under the control of the cauliflower mosaic virus 35S promoter were prepared. Transient expression assays in tobacco cell cultures and mung bean leaves showed that the VR-ACS1 and Cab22L 5'UTRs directed higher levels of GUS activity than the PAE 5'UTR. Analysis of transgenic Arabidopsis thaliana seedlings, as well as different tissues from mature plants, confirmed that while transcript levels were equivalent for all constructs, the 5'UTRs from the VR-ACS1 and Cab22L genes can increase GUS activity twofold to fivefold compared to the PAE 5'UTR, therefore confirming the translational enhancing properties of the VR-ACS1 5'UTR.

  12. Mechanism of translational regulation by miR-2 from sites in the 5′ untranslated region or the open reading frame

    PubMed Central

    Moretti, Francesca; Thermann, Rolf; Hentze, Matthias W.

    2010-01-01

    MicroRNAs (miRs) commonly regulate translation from target mRNA 3′ untranslated regions (UTRs). While effective miR-binding sites have also been identified in 5′ untranslated regions (UTRs) or open reading frames (ORFs), the mechanism(s) of miR-mediated regulation from these sites has not been defined. Here, we systematically investigate how the position of miR-binding sites influences translational regulation and characterize their mechanistic basis. We show that specific translational regulation is elicited in vitro and in vivo not only from the 3′UTR, but equally effectively from six Drosophila miR-2-binding sites in the 5′UTR or the ORF. In all cases, miR-2 triggers mRNA deadenylation and inhibits translation initiation in a cap-dependent fashion. In contrast, single or dual miR-2-binding sites in the 5′UTR or the ORF yield rather inefficient or no regulation. This work represents the first demonstration that 5′UTR and ORF miR-binding sites can function mechanistically similarly to the intensively investigated 3′UTR sites. Using single or dual binding sites, it also reveals a biological rationale for the high prevalence of miR regulatory sites in the 3′UTR. PMID:20966199

  13. Dis3- and exosome subunit-responsive 3 Prime mRNA instability elements

    SciTech Connect

    Kiss, Daniel L.; Hou, Dezhi; Gross, Robert H.; Andrulis, Erik D.

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. Black-Right-Pointing-Pointer Identified novel 3 Prime UTR cis-acting element that destabilizes a reporter mRNA. Black-Right-Pointing-Pointer Show exosome subunits are required for cis-acting element-mediated mRNA instability. Black-Right-Pointing-Pointer Define precise sequence requirements of novel cis-acting element. Black-Right-Pointing-Pointer Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3 Prime -5 Prime exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3 Prime untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are

  14. Regulated expression of nuclear protein(s) in myogenic cells that binds to a conserved 3' untranslated region in pro alpha 1 (I) collagen cDNA.

    PubMed Central

    Herget, T; Burba, M; Schmoll, M; Zimmermann, K; Starzinski-Powitz, A

    1989-01-01

    We describe the identification and DNA-binding properties of nuclear proteins from rat L6 myoblasts which recognize an interspecies conserved 3' untranslated segment of pro alpha 1 (I) collagen cDNA. Levels of the two pro alpha 1 (I) collagen RNAs, present in L6 myoblasts, decreased drastically between 54 and 75 h after induction of myotube formation in serum-free medium. Both mRNAs contained a conserved sequence segment of 135 nucleotides (termed tame sequence) in the 3' untranslated region that had 96% homology to the human and murine pro alpha 1 (I) collagen genes. The cDNA of this tame sequence was specifically recognized by nuclear protein(s) from L6 myoblasts, as judged by gel retardation assays and DNase I footprints. The tame-binding protein(s) was able to recognize its target sequence on double-stranded DNA but bound also to the appropriate single-stranded oligonucleotide. Protein that bound to the tame sequence was undetectable in nuclear extracts of L6 myotubes that did not accumulate the two collagen mRNAs. Therefore, the activity of this nuclear protein seems to be linked to accumulation of the sequences that it recognizes in vitro. The collagen RNAs and the nuclear tame-binding proteins reappeared after a change of medium, which further suggests that the RNAs and the protein(s) are coordinately regulated. Images PMID:2779548

  15. High genetic diversity in the coat protein and 3 untranslated regions among geographical isolates of Cardamom mosaic virus from south India.

    PubMed

    Jacob, T; Jebasingh, T; Venugopal, M N; Usha, R

    2003-09-01

    A survey was conducted to study the biological and genetic diversity of Cardamom mosaic virus (CdMV) that causes the most widespread disease in the cardamom growing area in the Western Ghats of south India. Six distinct subgroups were derived based on their symptomatology and host range from the sixty isolates collected. The serological variability between the virus isolates was analysed by ELISA and Western blotting. The 3 terminal region consisting of the coat protein (CP) coding sequence and 3 untranslated region (3 UTR) was cloned and sequenced from seven isolates. Sequence comparisons revealed considerable genetic diversity among the isolates in their CP and 3 UTR, making CdMV one of the highly variable members of Potyviridae. The possible occurrence of recombination between the isolates and the movement of the virus in the cardamom tract of south India are discussed.

  16. Polymorphisms in the 3′-untranslated region of the CDH1 gene are a risk factor for primary gastric diffuse large B-cell lymphoma

    PubMed Central

    Jacobs, Gunnar; Hellmig, Stephan; Huse, Klaus; Titz, Andrea; Franke, Andre; Kwiatkowski, Ruta; Ott, Stephan; Kosmahl, Markus; Fischbach, Wolfgang; Lucius, Ralph; Klapper, Wolfram; Fölsch, Ulrich R.; Hampe, Jochen; Schreiber, Stefan; Rosenstiel, Philip

    2011-01-01

    Background Primary gastric B-cell lymphomas arise from mucosa-associated lymphatic tissue (MALT) in patients with chronic Helicobacter pylori infection. We investigated whether germline variants in the CDH1 gene, coding for E-cadherin, genetically predispose patients to primary gastric B-cell lymphoma. Design and Methods Single marker analyses of the CDH1 gene were conducted in patients with primary gastric B-cell lymphoma (n=144), in patients with primary gastric high-grade lymphoma (n=61), and in healthy blood donors (n=361). Twelve single nucleotide polymorphisms were genotyped by TaqMan® technology. Allelic imbalance was tested by pyrosequencing and clone direct sequencing of heterozygote genomic and cDNA. Mutation detection was conducted around the poly-A signal of the CDH1 3′-untranslated region. The influence of the 3′-untranslated region on protein translation was determined by a luciferase reporter assay. Results Single marker analyses identified two single nucleotide polymorphisms in strong linkage disequilibrium located in the CDH1 3′-untranslated region. One of them was significantly associated with primary gastric diffuse large B-cell lymphomas after correction for multiple testing and this association was confirmed in an independent sample set. Patients homozygous for the rare T allele (rs1801026) had a 4.9-fold increased risk (95% CI: 1.5–15.9) of developing primary gastric diffuse large B-cell lymphoma. Allelic imbalance and reporter gene assays indicated a putative influence on mRNA stability and/or translational efficacy. Conclusions We identified variants in CDH1 as the first potential genetic risk factors for the development of primary gastric diffuse large B-cell lymphomas. One of the potentially causative variants affects allelic CDH1 expression. These findings support the hypothesis that besides somatic alterations of B-cells, germline variants in the CDH1 gene contribute to a predisposition to the development of primary gastric

  17. miR-155* mediates suppressive effect of PTEN 3'-untranslated region on AP-1/NF-κB pathway in HTR-8/SVneo cells.

    PubMed

    Xue, P; Zheng, M; Diao, Z; Shen, L; Liu, M; Gong, P; Sun, H; Hu, Y

    2013-08-01

    Among miRNAs, miR-155 is a known regulator of immune system. Accumulating studies have revealed the connections between miR-155 and activator protein 1 (AP-1)/nuclear factor (NF)-κB. However, miR-155*, a miR-155 paralog, has so far been less studied. Here we demonstrated that miR-155*, induced by lipopolysaccharide (LPS) in an AP-1/NF-κB dependent manner, played a positive feedback role in AP-1/NF-κB pathway via targeting interleukin-1 receptor-associated kinase M (IRAKM) and NF-κB inhibitor interacting Ras-like 1 (NKIRAS1) in trophoblasts. Our study further proved that miR-155*-targeted PTEN 3'-untranslated region (3'UTR) increased IRAKM and NKIRAS1 expression by competing for miR-155* binding, thereby suppressing AP-1/NF-κB activation induced by LPS.

  18. GB Virus C/Hepatitis G Virus Groups and Subgroups: Classification by a Restriction Fragment Length Polymorphism Method Based on Phylogenetic Analysis of the 5′ Untranslated Region

    PubMed Central

    Quarleri, J. F.; Mathet, V. L.; Feld, M.; Ferrario, D.; della Latta, M. P.; Verdun, R.; Sánchez, D. O.; Oubiña, J. R.

    1999-01-01

    A phylogenetic tree based on 150 5′ untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that HinfI and either AatII or AciI could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out. PMID:10203483

  19. RNA structure disrupting G320-T transversion within the short fragment of the 5' untranslated region prevents rescue of infectious foot-and-mouth disease virus.

    PubMed

    Mohapatra, Jajati K; Pandey, Laxmi K; Pattnaik, Bramhadev

    2014-02-01

    A full-length cDNA clone of an Indian foot-and-mouth disease virus strain, Asia 1 IND 491/1997 was assembled downstream of the T7 promoter in the pBluescript II SK (+) vector by sequential ligation of four PCR-generated subgenomic fragments. RNA transcribed from that construct were transfected into BHK-21 and LFBK cells to rescue infectious virus. The in vitro growth kinetics, plaque morphology, infectivity titer, antigenic profile and virulence characteristics in unweaned mice infected with the recombinant virus were comparable to those infected with the parental virus. However, repeated attempts to recover viable virus from the RNA transcripts with a G320-T point mutation introduced in the short fragment of the 5' untranslated region failed. The possible destabilizing effect of such a mutation on the predicted long stem-loop structure at the 5'-end of the genome and its implications for viral genome replication are discussed.

  20. Linkage disequilibrium between polymorphisms at the 5{prime} untranslated region and intron 5 (Dde I) of the antithrombin III (ATIII) gene in the Chinese

    SciTech Connect

    Tay, J.S.H.; Liu, Y.; Low, P.S.

    1994-09-01

    A length polymorphism at the 5{prime} untranslated region of exon 1 and an RFLP (Dde I) in intron 5 (nt 160) of the ATIII gene were amplified by polymerase chain reaction with primers of published sequences. DNA fragments were size-fractionated by agarose gel electrophoresis (3% NuSieve and 1% Seakem GTG) and photographed over a UV transilluminator. A strong linkage disequilibrium was observed between these two polymorphisms of the ATIII gene in the Chinese ({chi}{sup 2} = 63.7; {triangle} 0.42, P < 0.001). The estimated frequencies of the three haplotypes were found to be 0.37 for SD+, 0.40 for LD+ and 0.23 for LD-.

  1. Light regulation of Fed-1 mRNA requires an element in the 5' untranslated region and correlates with differential polyribosome association.

    PubMed Central

    Dickey, L F; Petracek, M E; Nguyen, T T; Hansen, E R; Thompson, W F

    1998-01-01

    Light regulation of Fed-1 mRNA abundance in the leaves of green plants is primarily a post-transcriptional process. Previously, we have shown that the Fed-1 mRNA light response requires an open reading frame, indicating that the light regulation of the mRNA depends on its concurrent translation. We now show that light-induced increases in Fed-1 mRNA abundance are associated with increases in polyribosome association that require both a functional AUG and a normal Fed-1 translational start context. We also present evidence that light regulation of Fed-1 mRNA levels requires more than efficient translation per se. Substitution of the efficiently translated tobacco mosaic virus Omega 5' untranslated region resulted in a loss of Fed-1 light regulation. In addition, we identified a CAT T repeat element located near the 5' terminus of the Fed-1 5' untranslated region that is essential for light regulation. We introduced two different mutations in the CAT T repeat element, but only one of these substitutions blocked the normal light effect on polyribosome association, whereas both altered dark-induced Fed-1 mRNA disappearance. The element may thus be important for Fed-1 mRNA stability rather than polyribosome loading. We propose a model in which Fed-1 mRNA is relatively stable when it is associated with polyribosomes in illuminated plants but in darkness is not polyribosome associated and is thus rapidly degraded by a process involving the CAT T repeat element. PMID:9501119

  2. Untranslated leader region polymorphism of Tvv1, a retrotransposon family, is a novel marker useful for analyzing genetic diversity and relatedness in the genus Vitis.

    PubMed

    Pelsy, Frédérique

    2007-12-01

    Grapevine retrotransposons belonging to the Tvv1 family share a single, highly conserved open reading frame but differ by their untranslated leader (UTL) region, which is highly variable in size. Amplification of the UTL region of Tvv1 elements from 94 Vitaceae accessions reveals that each of them shows a unique pattern of UTL-derived bands, which is inherited in progenies but conserved between clones vegetatively propagated. The overall organization of genetic diversity of the Vitaceae at the inter and intraspecific level and relatedness among accessions described by UTL-derived bands was compared to those obtained using 15 microsatellite loci. Both fingerprinting methods show a similar grouping of Vitis vinifera accessions but UTL-based fingerprinting more accurately isolates the muscadine grapes from the American and Asian Vitis. Finally, sequence analysis of seven UTL regions determines that their size variation is essentially caused by large deletions/insertions within the internal region, whereas flanking regions are more conserved. UTL-based fingerprinting could be considered as a novel marker system specific of the genus Vitis; moreover, as this multiband genotype is stable between clones it is suitable to be used as a "DNA barcode" for Vitis identification.

  3. Sequence analysis of the 3'-untranslated region of HSP70 (type I) genes in the genus Leishmania: its usefulness as a molecular marker for species identification.

    PubMed

    Requena, Jose M; Chicharro, Carmen; García, Lineth; Parrado, Rudy; Puerta, Concepción J; Cañavate, Carmen

    2012-04-28

    The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. In the present study, we analyzed the 3'-untranslated region (UTR) of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. It was observed that there was a remarkable degree of sequence conservation in this region, even between species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3'-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex) typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. Sequence and phylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination.

  4. Sequence analysis of the 3’-untranslated region of HSP70 (type I) genes in the genus Leishmania: its usefulness as a molecular marker for species identification

    PubMed Central

    2012-01-01

    Background The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. Methods In the present study, we analyzed the 3’-untranslated region (UTR) of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. Results It was observed that there was a remarkable degree of sequence conservation in this region, even between species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3´-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex) typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. Conclusions Sequence and phylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination. PMID:22541251

  5. Genome-wide association identifies a deletion in the 3' untranslated region of striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy.

    PubMed

    Meurs, Kathryn M; Mauceli, Evan; Lahmers, Sunshine; Acland, Gregory M; White, Stephen N; Lindblad-Toh, Kerstin

    2010-09-01

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins. We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine mapping and direct DNA sequencing identified an 8-bp deletion in the 3' untranslated region (UTR) of the Striatin gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3' UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in Striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized Striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, Plakophilin-2, Plakoglobin and Desmoplakin, all involved in the pathogenesis of ARVC in human beings. We suggest that Striatin may serve as a novel candidate gene for human ARVC.

  6. The 5' untranslated region of the rbp1 mRNA is required for translation of its mRNA under low temperatures in the cyanobacterium Synechococcus elongatus.

    PubMed

    Hayashi, Rie; Sugita, Chieko; Sugita, Mamoru

    2017-01-01

    The unicellular cyanobacterium Synechococcus elongatus has three RNA-binding protein (Rbp) genes, rbp1, rbp2 and rbp3. The rbp1 gene was upregulated by cold treatment while rbp2 and rbp3 expression decreased remarkably after exposure to cold temperatures. To investigate the mechanism underlying cold-induced rbp1 expression, a series of rbp1-luxAB transcriptional fusion constructs were expressed in S. elongatus PCC 7942 under cold conditions. The results showed that the region from -33 to -3 of the transcription initiation site contains an essential sequence for basal transcription of the rbp1 gene and that the 120-bp region (-34 to -153) does not contain critical cis-elements required for cold-shock induction. In contrast, mutational analysis carrying the 5'-untranslated region (UTR) of rbp1-luxAB translational fusions indicated that the 5'-UTR of rbp1 plays an important role in cold induction of the rbp1 gene product. Taken together, we conclude that the cold induction of rbp1 may be regulated at a posttranscriptional level rather than at the transcriptional level.

  7. Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

    PubMed

    Gritsun, Dmitri J; Jones, Ian M; Gould, Ernest A; Gritsun, Tamara S

    2014-01-01

    RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

  8. Complex Effects of Deletions in the 5′ Untranslated Region of Primate Foamy Virus on Viral Gene Expression and RNA Packaging

    PubMed Central

    Heinkelein, Martin; Thurow, Jana; Dressler, Marco; Imrich, Horst; Neumann-Haefelin, Dieter; McClure, Myra O.; Rethwilm, Axel

    2000-01-01

    Due to various advantageous features there is current interest in retroviral vectors derived from primate foamy viruses (PFVs). Two PFV cis-acting sequences have been mapped in the 5′ region of the RNA (pre-)genome and in the 3′ pol genomic region. In order to genetically separate PFV packaging constructs from vector constructs, we investigated the effect of deletions in the 5′ untranslated region (UTR) of PFV packaging constructs and vectors on gene expression and RNA incorporation into viral particles. Our results indicate that the 5′ UTR serves different previously unknown functions. First, the R region of the long terminal repeat was found to be required for PFV gag gene expression. This regulation of gene expression appeared to be mainly posttranscriptional. Second, constructs with sequence deletions between the R region and the gag gene start codon packaged as much viral mRNA into particles as the undeleted construct, and RNA from such a 5′-UTR-deleted packaging construct was copackaged into vector-virus particles, together with vector RNA which was preferentialy packaged. Finally, in the U5 region a sequence was identified that was required to allow cleavage of the Gag precursor protein by the pol gene-encoded protease, suggesting a role of RNA in PFV particle formation. Taken together, the results indicate that complex interactions of the viral RNA, capsid, and polymerase proteins take place during PFV particle formation and that a clear separation of PFV vector and packaging construct sequences may be difficult to achieve. PMID:10708430

  9. MicroRNA-296 is enriched in cancer cells and downregulates p21WAF1 mRNA expression via interaction with its 3' untranslated region.

    PubMed

    Yoon, A-rum; Gao, Ran; Kaul, Zeenia; Choi, Il-Kyu; Ryu, Jihoon; Noble, Jane R; Kato, Yoshio; Saito, Soichiro; Hirano, Takashi; Ishii, Tetsuro; Reddel, Roger R; Yun, Chae-Ok; Kaul, Sunil C; Wadhwa, Renu

    2011-10-01

    MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.

  10. Evaluation of a new-generation line-probe assay that detects 5' untranslated and core regions to genotype and subtype hepatitis C virus.

    PubMed

    Nadarajah, Rohan; Khan, G Yasmeen; Miller, Steven A; Brooks, Geo F

    2007-08-01

    The VERSANT HCV Genotype 2.0 Assay (LiPA 2.0; Innogenetics, Ghent, Belgium; distributed by Siemens Medical Solutions Diagnostics, Tarrytown, NY) is a new-generation line-probe assay that simultaneously detects sequences in the 5' untranslated (5'UTR) and core regions to genotype and subtype hepatitis C virus (HCV). We tested 60 specimens of known genotype and subtype and 2 specimens with mixed infections with the LiPA 2.0 assay. After arbitration based on genotype and subtype determined by sequencing, there was concordance in 58 of 60 specimens (specificity, 96.7%). Computer-assisted typing yielded comparable results, but much more rapidly. Of 67 clinical specimens, 64 readily yielded genotype and subtype; 3 indeterminate specimens were typed by sequencing and were uncommon types not in the database. The newgeneration line-probe assay that detects the 5'UTR and core regions to genotype and subtype HCV is applicable to more than 95% of specimens. Interpretation is facilitated by computer-assisted analysis.

  11. Target specificity of neuronal RNA-binding protein, Mel-N1: direct binding to the 3' untranslated region of its own mRNA.

    PubMed Central

    Abe, R; Yamamoto, K; Sakamoto, H

    1996-01-01

    We have identified cDNAs encoding Mel-N1, the mouse homologue of a human nervous system-specific RNA-binding protein, Hel-N1. Two major mRNA transcripts of Mel-N1 were detected predominantly in the adult mouse brain by Northern blot analysis. To gain insight into the RNA binding specificity of Mel-N1, we performed iterative in vitro RNA selection. The resulting in vitro selected RNAs were found to contain AU-rich sequences as well as a GAAA motif in the majority of clones. By means of in vitro binding assays we demonstrate that this GAAA sequence appears to significantly affect the Mel-N1 RNA-binding efficiency. Our studies further reveal that Mel-N1 can bind to its own 3' untranslated region (3'UTR) as well as to the c-fos 3'UTR, and is localized predominantly in the cytoplasmic region in cells, suggesting that posttranscriptional autoregulation of Mel-N1 gene expression occurs in vivo. PMID:8668530

  12. Correlation of the 5'untranslated region (5'UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping.

    PubMed

    Baclig, Michael O; Chan, Veronica F; Ramos, John Donnie A; Gopez-Cervantes, Juliet; Natividad, Filipinas F

    2010-07-07

    The 5'untranslated region (5'UTR) is often targeted to detect major genotypes in hepatitis C virus (HCV) but its insufficient sequence variation limits its usefulness for differentiating HCV subtypes. Subtyping has important implications to epidemiologic studies, clinical management, and vaccine development. Analysis of the nucleotide sequence of variable regions such as the non-structural 5B (NS5B) is considered the reference method for identifying HCV subtypes. We evaluated the accuracy of subtyping of HCV genotype 1 (HCV-1) samples from the Philippines by 5'UTR sequencing as compared with the NS5B sequence. A total of 30 patients infected with HCV-1 previously confirmed by PCR-RFLP and clinically diagnosed with chronic hepatitis C were analyzed. Nucleotide sequencing of the 5'UTR showed that 15 (50%) were identified as 1a and 15 (50%) were identified as 1b. Sequence analysis of the NS5B revealed that 13 (43%) belonged to subtype 1a while 17 (57%) belonged to subtype 1b. The most predominant subtype was 1b by NS5B sequencing. The predictive value of 5'UTR sequencing to subtype 1a was 73% while for subtype 1b, predictive value was 87%. Overall concordance between 5'UTR and NS5B sequencing was 80%. NS5B sequence and phylogenetic analysis is still the reference method for identifying HCV-1a and 1b subtypes.

  13. Correlation of the 5′untranslated region (5′UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping

    PubMed Central

    Baclig, Michael O; Chan, Veronica F; Ramos, John Donnie A; Gopez-Cervantes, Juliet; Natividad, Filipinas F

    2010-01-01

    The 5′untranslated region (5′UTR) is often targeted to detect major genotypes in hepatitis C virus (HCV) but its insufficient sequence variation limits its usefulness for differentiating HCV subtypes. Subtyping has important implications to epidemiologic studies, clinical management, and vaccine development. Analysis of the nucleotide sequence of variable regions such as the non-structural 5B (NS5B) is considered the reference method for identifying HCV subtypes. We evaluated the accuracy of subtyping of HCV genotype 1 (HCV-1) samples from the Philippines by 5′UTR sequencing as compared with the NS5B sequence. A total of 30 patients infected with HCV-1 previously confirmed by PCR-RFLP and clinically diagnosed with chronic hepatitis C were analyzed. Nucleotide sequencing of the 5′UTR showed that 15 (50%) were identified as 1a and 15 (50%) were identified as 1b. Sequence analysis of the NS5B revealed that 13 (43%) belonged to subtype 1a while 17 (57%) belonged to subtype 1b. The most predominant subtype was 1b by NS5B sequencing. The predictive value of 5′UTR sequencing to subtype 1a was 73% while for subtype 1b, predictive value was 87%. Overall concordance between 5′UTR and NS5B sequencing was 80%. NS5B sequence and phylogenetic analysis is still the reference method for identifying HCV-1a and 1b subtypes. PMID:21537395

  14. Functional equivalence of common and unique sequences in the 3' untranslated regions of alfalfa mosaic virus RNAs 1, 2, and 3.

    PubMed Central

    van Rossum, C M; Brederode, F T; Neeleman, L; Bol, J F

    1997-01-01

    The 3' untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3'-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3' UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3'-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding. PMID:9094656

  15. A Single Nucleotide in Stem Loop II of 5′-Untranslated Region Contributes to Virulence of Enterovirus 71 in Mice

    PubMed Central

    Yeh, Ming-Te; Wang, Shainn-Wei; Yu, Chun-Keung; Lin, Kuei-Hsiang; Lei, Huan-Yao; Su, Ih-Jen; Wang, Jen-Ren

    2011-01-01

    Background Enterovirus 71 (EV71) has emerged as a neuroinvasive virus responsible for several large outbreaks in the Asia-Pacific region while virulence determinant remains unexplored. Principal Findings In this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5′-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5′-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5′-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo. Conclusions These results presented the first reported virulence determinant in EV71 5′-UTR and first position discovered from unadapted isolates. PMID:22069490

  16. Ultra-Deep Sequencing Analysis of the Hepatitis A Virus 5'-Untranslated Region among Cases of the Same Outbreak from a Single Source

    PubMed Central

    Wu, Shuang; Nakamoto, Shingo; Kanda, Tatsuo; Jiang, Xia; Nakamura, Masato; Miyamura, Tatsuo; Shirasawa, Hiroshi; Sugiura, Nobuyuki; Takahashi-Nakaguchi, Azusa; Gonoi, Tohru; Yokosuka, Osamu

    2014-01-01

    Hepatitis A virus (HAV) is a causative agent of acute viral hepatitis for which an effective vaccine has been developed. Here we describe ultra-deep pyrosequences (UDPSs) of HAV 5'-untranslated region (5'UTR) among cases of the same outbreak, which arose from a single source, associated with a revolving sushi bar. We determined the reference sequence from HAV-derived clone from an attendant by the Sanger method. Sixteen UDPSs from this outbreak and one from another sporadic case were compared with this reference. Nucleotide errors yielded a UDPS error rate of < 1%. This study confirmed that nucleotide substitutions of this region are transition mutations in outbreak cases, that insertion was observed only in non-severe cases, and that these nucleotide substitutions were different from those of the sporadic case. Analysis of UDPSs detected low-prevalence HAV variations in 5'UTR, but no specific mutations associated with severity in these outbreak cases. To our surprise, HAV strains in this outbreak conserved HAV IRES sequence even if we performed analysis of UDPSs. UDPS analysis of HAV 5'UTR gave us no association between the disease severity of hepatitis A and HAV 5'UTR substitutions. It might be more interesting to perform ultra-deep sequencing of full length HAV genome in order to reveal possible unknown genomic determinants associated with disease severity. Further studies will be needed. PMID:24396287

  17. Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

    PubMed Central

    Staley, Chris A.; Huang, Amy; Nattestad, Maria; Oshiro, Kristin T.; Ray, Laura E.; Mulye, Tejas; Li, Zhiguo Harry; Le, Thu; Stephens, Justin J.; Gomez, Seth R.; Moy, Allison D.; Nguyen, Jackson C.; Franz, Andreas H.; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P.

    2012-01-01

    Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5′ untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5′UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5′UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5′UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5′UTR optimized for a higher level of expression may be challenging. PMID:22285974

  18. Interactome analysis of the EV71 5' untranslated region in differentiated neuronal cells SH-SY5Y and regulatory role of FBP3 in viral replication.

    PubMed

    Huang, Hsing-I; Chang, Ying-Ying; Lin, Jhao-Yin; Kuo, Rei-Lin; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-09-01

    Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia-Pacific region. Through interactions with host proteins, the 5' untranslated region (5'UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5'UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC-MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein-protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind to the EV71 5'UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5'UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.

  19. An Indel Polymorphism in the MtnA 3' Untranslated Region Is Associated with Gene Expression Variation and Local Adaptation in Drosophila melanogaster

    PubMed Central

    Glaser-Schmitt, Amanda; Duchen, Pablo; Parsch, John

    2016-01-01

    Insertions and deletions (indels) are a major source of genetic variation within species and may result in functional changes to coding or regulatory sequences. In this study we report that an indel polymorphism in the 3’ untranslated region (UTR) of the metallothionein gene MtnA is associated with gene expression variation in natural populations of Drosophila melanogaster. A derived allele of MtnA with a 49-bp deletion in the 3' UTR segregates at high frequency in populations outside of sub-Saharan Africa. The frequency of the deletion increases with latitude across multiple continents and approaches 100% in northern Europe. Flies with the deletion have more than 4-fold higher MtnA expression than flies with the ancestral sequence. Using reporter gene constructs in transgenic flies, we show that the 3' UTR deletion significantly contributes to the observed expression difference. Population genetic analyses uncovered signatures of a selective sweep in the MtnA region within populations from northern Europe. We also find that the 3’ UTR deletion is associated with increased oxidative stress tolerance. These results suggest that the 3' UTR deletion has been a target of selection for its ability to confer increased levels of MtnA expression in northern European populations, likely due to a local adaptive advantage of increased oxidative stress tolerance. PMID:27120580

  20. Haplotypes of the HLA-G 3' Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially.

    PubMed

    Poras, Isabelle; Yaghi, Layale; Martelli-Palomino, Gustavo; Mendes-Junior, Celso T; Muniz, Yara Costa Netto; Cagnin, Natalia F; Sgorla de Almeida, Bibiana; Castelli, Erick C; Carosella, Edgardo D; Donadi, Eduardo A; Moreau, Philippe

    2017-01-01

    The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5' and 3' regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3' untranslated region (3'UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3'UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3'UTR haplotypes in healthy individuals and reinforce that 3'UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G.

  1. Haplotypes of the HLA-G 3’ Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially

    PubMed Central

    Cagnin, Natalia F.; Sgorla de Almeida, Bibiana; Castelli, Erick C.; Carosella, Edgardo D.; Donadi, Eduardo A.; Moreau, Philippe

    2017-01-01

    The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5’ and 3’ regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3’ untranslated region (3’UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3’UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3’UTR haplotypes in healthy individuals and reinforce that 3’UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G. PMID:28045999

  2. Characterization of the partial RNA1 and RNA2 3' untranslated region of tomato ringspot virus isolates from North America

    USDA-ARS?s Scientific Manuscript database

    The 3' non-translated regions (NTRs) of RNA1 and RNA2 of Tomato ringspot virus (ToRSV) are long and virtually identical. In this study, sequences containing most of the 3’ NTRs (1168-1265 bp) were determined from 18 ToRSV isolates collected from fruit trees, small fruits, and grapevines in North Am...

  3. Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.

    PubMed

    Barbagallo, Michael S; Birch, Katherine E; Deacon, Nicholas J; Mosse, Jennifer A

    2012-07-01

    The negative-sense asp open reading frame (ORF) positioned opposite to the human immunodeficiency virus type 1 (HIV-1) env gene encodes the 189 amino acid, membrane-associated ASP protein. Negative-sense transcription, regulated by long terminal repeat sequences, has been observed early in HIV-1 infection in vitro. All subtypes of HIV-1 were scanned to detect the negative-sense asp ORF and to identify potential regulatory sequences. A series of highly conserved upstream short open reading frames (sORFs) was identified. This potential control region from HIV-1(NL4-3), containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant changes in levels of EGFP mRNA. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product is spliced to remove sORFs I to V, leaving only the in-frame sORF VI upstream of asp. Sequence analysis revealed the presence of typical splice donor- and acceptor-site motifs. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of EGFP production. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1. Alternative splicing of the upstream sORF region provides a potential mechanism for controlling expression of the asp gene.

  4. Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

    PubMed Central

    Wong, S C; Abdelal, A T

    1990-01-01

    The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated. Images FIG. 2 FIG. 3 FIG. 7 FIG. 8 PMID:2153657

  5. Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication

    PubMed Central

    Kawamura-Nagaya, Kazue; Ishibashi, Kazuhiro; Huang, Ying-Ping; Miyashita, Shuhei; Ishikawa, Masayuki

    2014-01-01

    Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided. PMID:24711385

  6. Expression of Trypanosoma cruzi surface antigen FL-160 is controlled by elements in the 3' untranslated, the 3' intergenic, and the coding regions.

    PubMed

    Weston, D; La Flamme, A C; Van Voorhis, W C

    1999-07-30

    The FL-160 surface antigen gene family of T. cruzi consists of hundreds of members of 160 kDa glycoproteins expressed in trypomastigotes, but not in epimastigotes. Steady-state levels of FL-160 mRNA were 80 to 100-fold higher in trypomastigotes than in epimastigotes, yet transcription rates were equivalent between the lifecycle stages. Luciferase reporter constructs demonstrated that the 3' untranslated region (UTR) and intergenic region (IR) following the coding sequence of FL-160 was sufficient to generate 8-fold higher luciferase expression in trypomastigotes compared with epimastigotes. Transfection of 3' UTR/IR deletion constructs revealed cis-acting elements which conferred a trypomastigote-specific expression pattern similar to that of FL-160. Parasites treated with translation and transcription inhibitors, cyclohexamide and Actinomycin D, respectively, displayed a stage-specific pattern of FL-160 mRNA degradation. Epimastigotes, but not trypomastigotes, treated with the inhibitors accumulated a 1.4 Kb FL-160 cleavage product. The cleavage site mapped to a 31 base poly-purine tract in the FL-160 coding region. The first 526 aa of FL-160, containing the 31 base poly-purine tract and several smaller tracts, were fused to green fluorescent protein (GFP) and expressed from the T. cruzi tubulin locus. Stable transformants expressed 4-fold more FL-160:GFP fusion mRNA and 12-fold more fusion protein in the trypomastigote stage than in the epimastigote stage suggesting post-transcriptional and translational control elements. These data reveal at least two distinct control mechanisms for trypomastigote-specific expression of FL-160 surface glycoproteins, one involving the 3' UTR/IR and one involving the coding region of FL-160.

  7. Ribosome binding to a 5' translational enhancer is altered in the presence of the 3' untranslated region in cap-independent translation of turnip crinkle virus.

    PubMed

    Stupina, Vera A; Yuan, Xuefeng; Meskauskas, Arturas; Dinman, Jonathan D; Simon, Anne E

    2011-05-01

    Plus-strand RNA viruses without 5' caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5' UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5' UTR sequences. Addition of 80S ribosomes to the 5' UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5' end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5'-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5' and 3' sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5' and 3' UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5' UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5' and 3' UTRs of TCV.

  8. Polymorphisms at the 3' untranslated region of SLC11A1 gene are associated with protection to Brucella infection in goats.

    PubMed

    Iacoboni, Paola A; Hasenauer, Flavia C; Caffaro, M Eugenia; Gaido, Analia; Rossetto, Cristina; Neumann, Roberto D; Salatin, Antonio; Bertoni, Emiliano; Poli, Mario A; Rossetti, Carlos A

    2014-08-15

    Goats are susceptible to brucellosis and the detection of Brucella-infected animals is carried out by serological tests. In other ruminant species, polymorphisms in microsatellites (Ms) of 3' untranslated region (3'UTR) of the solute carrier family 11 member A1 (SLC11A1) gene were associated with resistance to Brucella abortus infection. Goats present two polymorphic Ms at the 3'UTR end of SLC11A1 gene, called regions A and B. Here, we evaluated if polymorphisms in regions A and/or B are associated with Brucella infection in goats. Serum (for the detection of Brucella-specific antibodies) and hair samples (for DNA isolation and structure analysis of the SLC11A1 gene) were randomly collected from 229 adult native goats from the northwest of Argentina. Serological status was evaluated by buffer plate antigen test (BPAT) complemented by the fluorescent polarization assay (FPA), and the genotype of the 3'UTR of the SLC11A1 gene was determined by capillary electrophoresis and confirmed by sequence analysis. Polymorphisms in regions A and B of the 3'UTR SLC11A1 gene were found statistically significant associated with protection to Brucella infection. Specifically, the association study indicates statistical significance of the allele A15 and B7/B7 genotype with absence of Brucella-specific antibodies (p=0.0003 and 0.0088, respectively). These data open a promising opportunity for limiting goat brucellosis through selective breeding of animals based on genetic markers associated with natural resistance to B. melitensis infection.

  9. Endothelin-1 expression is strongly repressed by AU-rich elements in the 3′-untranslated region of the gene

    PubMed Central

    2004-01-01

    The regulation of the synthesis of the endothelial-derived vasoconstrictor ET-1 (endothelin-1) is a complex process that occurs mainly at the mRNA level. Transcription of the gene accounts for an important part of the regulation of expression, as already described for different modulators such as the cytokine TGF-β (transforming growth factor-β). However, very little is known about mechanisms governing ET-1 expression at the post-transcriptional level. The aim of the present study was to investigate the regulation of the ET-1 expression at this level. Since the 3′-UTR (3′-untranslated region) of mRNAs commonly contains genetic determinants for the post-transcriptional control of gene expression, we focused on the potential role of the 3′-UTR of ET-1 mRNA. Experiments performed with luciferase reporter constructs containing the 3′-UTR showed that this region exerts a potent destabilizing effect. Deletional analyses allowed us to locate this activity within a region at positions 924–1127. Some (but not all) of the AREs (AU-rich elements) present in this region were found to be essential for this mRNA-destabilizing activity. We also present evidence that cytosolic proteins from endothelial cells interact specifically with these RNA elements, and that a close correlation exists between the ability of the AREs to destabilize ET-1 mRNA and the binding of proteins to these elements. Our results are compatible with the existence of a strong repressional control of ET-1 expression mediated by destabilization of the mRNA exerted through the interaction of specific cytosolic proteins with AREs present in the 3′-UTR of the gene. PMID:15595926

  10. Targeting deoxyhypusine hydroxylase activity impairs cap-independent translation initiation driven by the 5'untranslated region of the HIV-1, HTLV-1, and MMTV mRNAs.

    PubMed

    Cáceres, C Joaquín; Angulo, Jenniffer; Contreras, Nataly; Pino, Karla; Vera-Otarola, Jorge; López-Lastra, Marcelo

    2016-10-01

    Replication of the human immunodeficiency virus type 1 (HIV-1) is dependent on eIF5A hypusination. Hypusine is formed post-translationally on the eIF5A precursor by two consecutive enzymatic steps; a reversible reaction involving the enzyme deoxyhypusine synthase (DHS) and an irreversible step involving the enzyme deoxyhypusine hydroxylase (DOHH). In this study we explored the effect of inhibiting DOHH activity and therefore eIF5A hypusination, on HIV-1 gene expression. Results show that the expression of proteins from an HIV-1 molecular clone is reduced when DOHH activity is inhibited by Deferiprone (DFP) or Ciclopirox (CPX). Next we evaluated the requirement of DOHH activity for internal ribosome entry site (IRES)-mediated translation initiation driven by the 5'untranslated region (5'UTR) of the full length HIV-1 mRNA. Results show that HIV-1 IRES activity relies on DOHH protein concentration and enzymatic activity. Similar results were obtained for IRES-dependent translation initiation mediated by 5'UTR of the human T-cell lymphotropic virus type 1 (HTLV-1) and the mouse mammary tumor virus (MMTV) mRNAs. Interestingly, activity of the poliovirus IRES, was less sensitive to the targeting of DOHH suggesting that not all viral IRESs are equally dependent on the cellular concentration or the activity of DOHH. In summary we present evidence indicating that the cellular concentration of DOHH and its enzymatic activity play a role in HIV-1, HTLV-1 and MMTV IRES-mediated translation initiation.

  11. The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

    PubMed Central

    Vallejos, Maricarmen; Ramdohr, Pablo; Valiente-Echeverría, Fernando; Tapia, Karla; Rodriguez, Felipe E.; Lowy, Fernando; Huidobro-Toro, J. Pablo; Dangerfield, John A.; López-Lastra, Marcelo

    2010-01-01

    In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5′-untranslated region (5′-UTR) of the mouse mammary tumor virus (MMTV). The 5′-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5′-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5′-UTR was resistant to the addition of m7GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5′-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function. PMID:19889724

  12. Involvement of the 5'-untranslated region in cold-regulated expression of the rbpA1 gene in the cyanobacterium Anabaena variabilis M3.

    PubMed Central

    Sato, N; Nakamura, A

    1998-01-01

    Transcript of the rbpA1 gene in Anabaena variabilis accumulates significantly at low growth temperatures below 28 degreesC. This accumulation was maximal at 16 degreesC. Accumulation of the rbpA1 transcript was completely abolished by rifampicin, but not by chloramphenicol. Photosynthesis was not required for this cold-induced accumulation. This accumulation of transcript was partly accounted for by increased stability of the rbpA1 transcript at low temperature. Expression of chimeric genes containing 3'-deleted rbpA1 sequences fused to the lacZ gene was regulated by low temperature when almost the entire 5'-untranslated region (5'-UTR) remained undeleted. Further deletion resulted in constitutive expression of the chimeric gene. The 5'-UTR sequence formed two types of complexes in vitro with protein extract from cells grown at 38 degreesC, but not with extract from the 22 degreesC grown cells. Affinity purification identified polypeptides of 75 and 32 kDa in Complex 1 and a 72 kDa polypeptide in Complex 2. These results are compatible with a model in which expression of the rbpA1 gene is regulated by transcriptional derepression at low temperature, although additional mechanisms, such as regulation of mRNA stability, might also contribute to temperature-dependent regulation. PMID:9547280

  13. The tRNA methyltransferase NSun2 stabilizes p16INK4 mRNA by methylating the 3′-untranslated region of p16

    PubMed Central

    Zhang, Xiaotian; Liu, Zhenyun; Yi, Jie; Tang, Hao; Xing, Junyue; Yu, Minqwei; Tong, Tanjun; Shang, Yongfeng; Gorospe, Myriam; Wang, Wengong

    2012-01-01

    The impact of methylation of the 3′-untranslated region (UTR) of a messenger RNA (mRNA) remains largely unknown. Here we show that NSun2, a transfer RNA methyltransferase, inhibits the turnover of p16INK4 mRNA. Knockdown of NSun2 reduces p16 expression by shortening the half-life of the p16 mRNA, while overexpression of NSun2 stabilizes the p16 mRNA. In vitro methylation assays show that NSun2 methylates the p16 3′UTR at A988. Knockdown of NSun2 reduces the stability of the EGFP-p16 chimeric reporter transcripts bearing wild-type p16 3′UTR, but not p16 3′UTR with a mutant methylation site. Methylation by NSun2 prevents the association of p16 3′UTR with HuR, AUF1 and Ago2/RISC, and prevents the recruitment of EGFP-p16 3′UTR chimeric transcripts to processing bodies. In response to oxidative stress, NSun2 is essential for elevating p16 expression levels. We conclude that NSun2-mediated methylation of the p16 3′UTR is a novel mechanism to stabilize p16 mRNA. PMID:22395603

  14. The tRNA methyltransferase NSun2 stabilizes p16INK⁴ mRNA by methylating the 3'-untranslated region of p16.

    PubMed

    Zhang, Xiaotian; Liu, Zhenyun; Yi, Jie; Tang, Hao; Xing, Junyue; Yu, Minqwei; Tong, Tanjun; Shang, Yongfeng; Gorospe, Myriam; Wang, Wengong

    2012-03-06

    The impact of methylation of the 3'-untranslated region (UTR) of a messenger RNA (mRNA) remains largely unknown. Here we show that NSun2, a transfer RNA methyltransferase, inhibits the turnover of p16(INK4) mRNA. Knockdown of NSun2 reduces p16 expression by shortening the half-life of the p16 mRNA, while overexpression of NSun2 stabilizes the p16 mRNA. In vitro methylation assays show that NSun2 methylates the p16 3'UTR at A988. Knockdown of NSun2 reduces the stability of the EGFP-p16 chimeric reporter transcripts bearing wild-type p16 3'UTR, but not p16 3'UTR with a mutant methylation site. Methylation by NSun2 prevents the association of p16 3'UTR with HuR, AUF1 and Ago2/RISC, and prevents the recruitment of EGFP-p16 3'UTR chimeric transcripts to processing bodies. In response to oxidative stress, NSun2 is essential for elevating p16 expression levels. We conclude that NSun2-mediated methylation of the p16 3'UTR is a novel mechanism to stabilize p16 mRNA.

  15. Alternative splicing within the elk-1 5' untranslated region serves to modulate initiation events downstream of the highly conserved upstream open reading frame 2.

    PubMed

    Rahim, Gwendoline; Araud, Tanguy; Jaquier-Gubler, Pascale; Curran, Joseph

    2012-05-01

    The 5' untranslated region (UTR) plays a central role in the regulation of mammalian translation initiation. Key components include RNA structure, upstream AUGs (uAUGs), upstream open reading frames (uORFs), and internal ribosome entry site elements that can interact to modulate the readout. We previously reported the characterization of two alternatively spliced 5' UTR isoforms of the human elk-1 gene. Both contain two uAUGs and a stable RNA stem-loop, but the long form (5' UTR(L)) was more repressive than the short form (5' UTR(S)) for initiation at the ELK-1 AUG. We now demonstrate that ELK-1 expression arises by a combination of leaky scanning and reinitiation, with the latter mediated by the small uORF2 conserved in both spliced isoforms. In HEK293T cells, a considerable fraction of ribosomes scans beyond the ELK-1 AUG in a reinitiation mode. These are sequestered by a series of out-of-frame AUG codons that serve to prevent access to a second in-frame AUG start site used to express short ELK-1 (sELK-1), an N-terminally truncated form of ELK-1 that has been observed only in neuronal cells. We present evidence that all these events are fine-tuned by the nature of the 5' UTR and the activity of the α subunit of eukaryotic initiation factor 2 and provide insights into the neuronal specificity of sELK-1 expression.

  16. Mosquito La protein binds to the 3' untranslated region of the positive and negative polarity dengue virus RNAs and relocates to the cytoplasm of infected cells.

    PubMed

    Yocupicio-Monroy, Martha; Padmanabhan, R; Medina, Fernando; del Angel, Rosa M

    2007-01-05

    The untranslated regions (UTRs) of the positive and negative strand RNAs of several viruses are major binding sites for cellular and viral proteins. Human La autoantigen is one of the cellular proteins that interacts with various positive strand RNA viral genomes including that of dengue virus (DEN) within the 5'- and 3'-UTRs of positive (+) and the 3'-UTR of negative strand (-) RNA, and with the nonstructural proteins NS3 and NS5, that form DEN replicase complex. Since DEN replicates in human and mosquito cells, some functional interactions have to be conserved in both hosts. In the present report, we demonstrate that mosquito La protein interacts with the 3'-UTRs of (+) and (-) polarity viral RNAs. The localization of La protein, examined by confocal microscopy, indicates that La protein is redistributed in DEN-infected cells. Furthermore, the presence of La protein in an in vitro replication system inhibited RNA synthesis in a dose-dependent manner, suggesting that La protein plays an important role in dengue virus replicative cycle.

  17. Increased nucleotide polymorphic changes in the 5′-untranslated region of δ-catenin (CTNND2) gene in prostate cancer

    PubMed Central

    Wang, Tao; Chen, Yan-Hua; Hong, Heng; Zeng, Yan; Zhang, Jiao; Lu, Jian-Ping; Jeansonne, Beverly; Lu, Qun

    2008-01-01

    Cancer pathogenesis involves multiple genetic and epigenetic alterations, which result in oncogenic changes in gene expression. δ-Catenin (CTNND2) is overexpressed in cancer although the mechanisms of its upregulation are highly variable. Here we report that in prostate cancer the methylation of CpG islands in δ-catenin promoter was not a primary regulatory event. There was also no δ-catenin gene amplification. However, using Single-Strand Conformation Polymorphism analysis, we observed the increased nucleotide changes in the 5′-untranslated region of δ-catenin gene in human prostate cancer. At least one such change (-9 G>A) is a true somatic point mutation associated with a high Gleason score, poorly differentiated prostatic adenocarcinoma. Laser capture microdissection coupled with PCR analyses detected the mutation only in cancerous but not in the adjacent benign prostatic tissues. Using chimeric genes encoding the luciferase reporter, we found that this mutation, but not a random mutation or a mutation that disrupts an upstream open reading frame, resulted in a remarkably higher expression and enzyme activity. This mutation did not affect transcriptional efficiency, suggesting that it promotes δ-catenin translation. This is the first report of δ-catenin gene mutation in cancer and supports the notion that multiple mechanisms contribute to its increased expression in carcinogenesis. PMID:18978817

  18. A Polymorphism in the 5′-Untranslated Region of the Porcine Cholecystokinin Type A Receptor Gene Affects Feed Intake and Growth

    PubMed Central

    Houston, R. D.; Haley, C. S.; Archibald, A. L.; Cameron, N. D.; Plastow, G. S.; Rance, K. A.

    2006-01-01

    The location and utilization of quantitative trait loci (QTL) and candidate genes with significant effects on economically important traits are becoming increasingly important in livestock breeding programs. The porcine cholecystokinin type A receptor (CCKAR) is a candidate gene for performance traits, due to its known role in the physiological control of feed intake, satiety, and obesity. We investigated the association of CCKAR polymorphisms with feeding, growth, and efficiency traits in an F2 population derived from a cross between Meishan and Large White founder animals and in lines of Large White pigs that had been divergently selected on the basis of lean growth efficiency traits. In the F2 population, CCKAR genotype was significantly associated with daily feed intake and average daily gain. The effects of the polymorphisms were then assessed in a larger-scale analysis of segregating commercial lines. A newly discovered single-nucleotide polymorphism (SNP) within the 5′-untranslated region (5′-UTR) had highly significant effects on feed intake, average daily gain, and days to 110 kg, which were not seen for a previously reported SNP within the CCKAR gene. Furthermore, we provide evidence that the novel SNP disrupts the binding of the YY1 transcription factor, which raises the possibility that it is the causal variant. The 5′-UTR SNP could be utilized as a molecular genetic test for increased feed intake, faster lean growth, and reduced days to market weight in segregating commercial lines. PMID:16951077

  19. A QPCR-based reporter system to study post-transcriptional regulation via the 3' untranslated region of mRNA in Saccharomyces cerevisiae.

    PubMed

    Lind, Kristina; Norbeck, Joakim

    2009-07-01

    Post-transcriptional regulation via the 3' untranslated region (3' UTR) of mRNA is an important factor in governing eukaryotic gene expression. Achieving detailed understanding of these processes requires highly quantitative systems in which comparative studies can be performed. To this end, we have developed a plasmid reporter system for Saccharomyces cerevisiae, in which the 3' UTR can be easily replaced and modified. Accurate quantification of the tandem affinity purification tag (TAP)-reporter protein and of TAP-mRNA is achieved by immuno-QPCR and by RT-QPCR, respectively. We have used our reporter system to evaluate the consequences on gene expression from varying the 3' UTR, a problem often encountered during C-terminal tagging of proteins. It was clear that the choice of 3' UTR was a strong determinant of the reporter expression, in a manner dependent on the growth conditions used. Mutations affecting either decapping (lsm1Delta) or deadenylation (pop2Delta) were also found to affect reporter gene expression in a highly 3' UTR-dependent manner. Our results using this set-up clearly indicate that the common strategy used for C-terminal tagging, with concomitant replacement of the native 3' UTR, will very likely provide incorrect conclusions on gene expression.

  20. Human tumor necrosis factor-alpha gene 3' untranslated region confers inducible toxin responsiveness to homologous promoter in monocytic THP-1 cells.

    PubMed

    Seiler-Tuyns, A; Dufour, N; Spertini, F

    1999-07-30

    To better define the role of 3' untranslated region (3'UTR) on transcriptional regulation of the human tumor necrosis factor (TNF)-alpha gene, monocytic human THP-1 cells were transfected with two TNF-alpha promoter constructs spanning base pairs -1897/-1 and -1214/-1, respectively, and linked to the rabbit beta-globin gene. Quantitative globin gene expression of chimerae was measured by reverse transcription-polymerase chain reaction. A construct linking the chicken beta-actin promoter and a deleted portion of the beta-globin gene was cotransfected and used as internal standard. Unexpectedly, when THP-1 cells were stimulated with lipopolysaccharide or toxic shock syndrome toxin-1, gene regulation was hardly detected. In contrast, endogenous TNF-alpha gene regulation measured by the same reverse transcription-polymerase chain reaction procedure was vigorous. Remarkably, ligation of 3'UTR to chimeric constructs led to a drastic drop in the basal level of chimeric gene expression, resulting in a 15- to 40-fold induction of the reporter gene. Consistently, when the TNF-alpha promoter was replaced by the cytomegalovirus early immediate promoter, gene expression was also uniformly reduced but was no longer up-regulated upon stimulation with lipopolysaccharide and toxic shock syndrome toxin-1. These data provide the first line of evidence that, in addition to its role in TNF-alpha transcript stability and translation, human TNF-alpha 3'UTR also participates in modulating gene expression at the transcriptional level.

  1. Efficient inhibition of human immunodeficiency virus replication using novel modified microRNA-30a targeting 3′-untranslated region transcripts

    PubMed Central

    NEJATI, AHMAD; SHAHMAHMOODI, SHOHREH; AREFIAN, EHSAN; SHOJA, ZABIHOLLAH; MARASHI, SAYED-MAHDI; TABATABAIE, HAMIDEH; MOLLAEI-KANDELOUS, YAGHOUB; SOLEIMANI, MASOUD; NATEGH, RAKHSHANDEH

    2016-01-01

    RNA interference (RNAi)-based gene therapy is currently considered to be a combinatorial anti-human immunodeficiency virus-1 (HIV-1) therapy. Although artificial polycistronic microRNAs (miRs) can reduce HIV-1 escape mutant variants, this approach may increase the risk of side effects. The present study aimed to optimize the efficiency of anti-HIV RNAi gene therapy in order to reduce the cell toxicity induced by multi-short hairpin RNA expression. An artificial miR-30a-3′-untranslated region (miR-3-UTR) obtained from a single RNA polymerase II was used to simultaneously target all viral transcripts. The results of the present study demonstrated that HIV-1 replication was significantly inhibited in the cells with the miR-3-UTR construct, suggesting that miR-3′-UTR may serve as a promising tool for RNAi-based gene therapy in the treatment of HIV-1. PMID:27168813

  2. The Effect of Poly(ADP-ribose) Polymerase-1 Gene 3'Untranslated Region Polymorphism in Colorectal Cancer Risk among Saudi Cohort.

    PubMed

    Alhadheq, Abdullah M; Purusottapatnam Shaik, Jilani; Alamri, Abdullah; Aljebreen, Abdulrahman M; Alharbi, Othman; Almadi, Majid A; Alhadeq, Faten; Azzam, Nahla A; Semlali, Abdelhabib; Alanazi, Mohammad; Bazzi, Mohammad D; Reddy Parine, Narasimha

    2016-01-01

    Background. DNA repair systems are essential for each cell to repair and maintain the genome integrity. Base excision repair pathway is one of the crucial pathways to maintain genome integrity and PARP-1 plays a key role in BER pathway. The purpose of this study is to evaluate the association between polymorphisms in PARP-1 3'untranslated region (3'UTR) SNP rs8679 and its expression in colorectal cancer. Methods. Genotyping and gene expression were performed using TaqMan assays. The effects of age, gender, and tumor location were evaluated in cases and controls regarding the genotyping results. Resulting data was analyzed using SPSS software. Results and Conclusions. Genotyping analysis for SNP rs8679 showed decreased susceptibility to colorectal cancer at heterozygous TC allele and at minor allele C. Further this protective association was also observed in younger age patients (≤57), in female patients, and also in patients with tumors located at colon and rectum. PARP-1 expression levels are significantly different in colorectal cancer compared to matched normal tissue. Our findings proved that the upregulation of PARP-1 is associated with tumor progression and poor prognosis in Saudi patients with colorectal cancer, suggesting that PARP-1 can be novel and valuable signatures for predicting the clinical outcome of patients with colorectal cancer.

  3. Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication.

    PubMed

    Cancio-Lonches, Clotilde; Yocupicio-Monroy, Martha; Sandoval-Jaime, Carlos; Galvan-Mendoza, Iván; Ureña, Luis; Vashist, Surender; Goodfellow, Ian; Salas-Benito, Juan; Gutiérrez-Escolano, Ana Lorena

    2011-08-01

    Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.

  4. Nucleolin Interacts with the Feline Calicivirus 3′ Untranslated Region and the Protease-Polymerase NS6 and NS7 Proteins, Playing a Role in Virus Replication ▿

    PubMed Central

    Cancio-Lonches, Clotilde; Yocupicio-Monroy, Martha; Sandoval-Jaime, Carlos; Galvan-Mendoza, Iván; Ureña, Luis; Vashist, Surender; Goodfellow, Ian; Salas-Benito, Juan; Gutiérrez-Escolano, Ana Lorena

    2011-01-01

    Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3′ untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication. PMID:21680514

  5. Transcriptional effects of a lupus-associated polymorphism in the 5´ untranslated region (UTR) of human complement receptor 2 (CR2/CD21)

    PubMed Central

    Cruickshank, Mark N.; Karimi, Mahdad; Mason, Rhonda L.; Fenwick, Emily; Mercer, Tim; Tsao, Betty P.; Boackle, Susan A.; Ulgiati, Daniela

    2012-01-01

    Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic component that determines risk. A common three single-nucleotide polymorphism (SNP) haplotype of the complement receptor 2 (CR2) gene has been associated with increased risk of SLE (Wu et al., 2007) (Douglas et al., 2009), and a less common haplotype consisting of the major allele at SNP1 and minor alleles at SNP2 and 3 confers protection (Douglas et al., 2009). SNP1 (rs3813946), which is located in the 5´ untranslated region (UTR) of the CR2 gene, altered transcriptional activity of a CR2 promoter-luciferase reporter gene construct transiently transfected into a B cell line (Wu et al., 2007) and had an independent effect in the protective haplotype (Douglas et al., 2009). In this study, we show that this SNP alters transcriptional activity in a transiently transfected non B-cell line as well as in stably transfected cell lines, supporting its relevance in vivo. Furthermore, the allele at this SNP affects chromatin accessibility of the surrounding sequence and transcription factor binding. These data confirm the effects of rs3813946 on CR2 transcription, identifying the 5´UTR to be a novel regulatory element for the CR2 gene in which variation may alter gene function and modify the development of lupus. PMID:22673213

  6. Synergistic roles of the E2 glycoprotein and 3' untranslated region in the increased genomic stability of chimeric classical swine fever virus with attenuated phenotypes.

    PubMed

    Wu, Rui; Li, Ling; Lei, Lei; Zhao, Cheng; Shen, Xiaofang; Zhao, Haizhong; Pan, Zishu

    2017-06-01

    The E2 glycoprotein and 3' untranslated region (UTR) of classical swine fever virus (CSFV) are virulence determinants. To investigate the synergistic roles of E2 and 3'UTR for pathogenicity and genomic stability, a series of chimeric CSFVs were constructed by replacing the E2 gene and/or 3'UTR of virulent CSFV strain Shimen with the corresponding sequence of the lapinized 'Chinese' strain (C-strain) using a reverse genetic approach. The in vitro growth characterization and in vivo pathogenicity of the chimeric CSFVs were investigated. Our results demonstrated that the E2 glycoprotein mediates virus cell-to-cell spread and viral particle release and that the 3'UTR regulates viral RNA replication. The CSFV E2 and 3'UTR synergistically modulate infectious virus production, viral genomic stability in vitro, and attenuation in swine. This work contributes to our understanding of the structure and function of the CSFV genome and virus pathogenicity and will be useful for the development of a novel CSF vaccine.

  7. Association of a miR-34b binding site single nucleotide polymorphism in the 3'-untranslated region of the methylenetetrahydrofolate reductase gene with susceptibility to male infertility.

    PubMed

    Zhang, W; Lin, W-Q; Cao, H-F; Li, C-Y; Li, F

    2015-10-09

    This study aims to explore the possible associations between a genetic variation in the miR-34b binding site in the 3'-untranslated region (UTR) of the methylenetetrahydrofolate reductase (MTHFR) gene (rs55763075) with male infertility in a Chinese population. Genotype distributions of the rs55763075 single nucleotide polymorphism were investigated by polymerase chain reaction and direct sequencing in a Chinese cohort that included 464 infertile men with idiopathic azoospermia or oligospermia and 458 controls with normal fertility. Overall, no significant differences in the distributions of the genotypes of the MTHFR rs55763075 polymorphism were detected between the infertility and control groups. A statistically significant increased risk of male infertility was found for carriers of the rs55763075 AA genotype when compared with homozygous carriers of the rs55763075 GG genotype in the azoospermia subgroup (OR = 1.721; 95% CI = 1.055-2.807; P = 0.031). Furthermore, we found that rs55763075 was associated with folate and homocysteine levels in patients with idiopathic azoospermia. Our results indicated that the MTHFR 3'-UTR rs55763075 polymorphism might modify the susceptibility to male infertility with idiopathic azoospermia.

  8. Inhibition of porcine reproductive and respiratory syndrome virus replication with exosome-transferred artificial microRNA targeting the 3' untranslated region.

    PubMed

    Zhu, Li; Bao, Liping; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2015-10-01

    Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease. As part of the development of RNA interference (RNAi) strategy against the disease, in this study a recombinant adenovirus (rAd) expressing the artificial microRNA (amiRNA) targeting the 3' untranslated region (UTR) was used to investigate the exosome-mediated amiRNA transfer from different pig cell types to porcine alveolar macrophages (PAMs). Quantitative RT-PCR showed that the sequence-specific amiRNA was expressed in and secreted via exosomes from the rAd-transduced pig kidney cell line PK-15, PAM cell line 3D4/163, kidney fibroblast cells (PFCs) and endometrial endothelial cells (PEECs) with different secretion efficiencies. Fluorescent microscopy revealed that the dye-labeled amiRNA-containing exosomes of different cell origins were efficiently taken up by all of the five types of pig cells tested, including primary PAMs. Quantitative RT-PCR showed that the amiRNA-containing exosomes of different cell origins were taken up by primary PAMs in both time- and dose-dependent manners. Both quantitative RT-PCR and viral titration assays showed that the exosome-delivered amiRNA had potent anti-viral effects against three different PRRSV strains. These data suggest that the exosomes derived from pig cells could serve as an efficient miRNA transfer vehicle, and that the exosome-delivered amiRNA had potent anti-viral effects against different PRRSV strains.

  9. Genetic polymorphism of 3' untranslated region of zeta-chain associated protein kinase 70 kDa in southern Taiwanese patients with rheumatoid arthritis.

    PubMed

    Chen, Shih-Yao; Liu, Ming-Fei; Wang, Chrong-Reen

    2016-03-01

    T cell activation participates in the pathogenesis of rheumatoid arthritis (RA), and the signaling molecule zeta-chain-associated protein kinase 70 kDa (ZAP-70) plays a crucial role in this process. Different mutations in the coding sequence of ZAP-70 are involved in a variety of immunological phenotypes, and recent evidence indicates that genetic variations within the 3' untranslated regions (UTR) of microRNA binding sites may affect the hybridization with target mRNAs, leading to phenotype changes with disease status. In this study, we evaluated the possible effect of ZAP-70 polymorphism as a genetic risk factor in RA by examining the single-nucleotide polymorphism in 100 patients and 100 ethnicity- and sex-matched healthy individuals from southern Taiwan. In both groups, the genotype distribution of rs2278699 in the 3' UTR was in the Hardy-Weinberg equilibrium. In RA, there were higher frequencies of the G allele (15.5 versus 8.0 %, odds ratio 2.1, P = 0.020) and significant differences in the trend of various genotypes (P = 0.024). The results suggest that genetic polymorphism in the 3' UTR of ZAP-70 is associated with RA susceptibility in southern Taiwanese.

  10. The 5' untranslated mRNA region base content can greatly affect translation initiation in the absence of secondary structures in Prevotella bryantii TC1-1.

    PubMed

    Seničar, Lenart; Accetto, Tomaž

    2015-01-01

    It has become clear lately that many bacteria and even whole bacterial phyla do not use the classical Shine-Dalgarno sequence mediated pathway of protein translation initiation. The prominent phylum Bacteroidetes is one of them, and this was shown not only using bioinformatic but also functional reporter gene studies in its representative Prevotella bryantii. The latter studies revealed much higher sensitivity toward secondary structures in 5(') untranslated mRNA regions (5(') UTRs) during translation initiation compared to Escherichia coli. It was proposed that in the absence of Shine-Dalgarno sequence interaction the key elements enabling translation initiation are local absence of secondary structures in 5(') UTRs, and the ribosomal protein S1 which binds to mRNA. Here, we evaluate the 5(') UTRs devoid of secondary structures but containing divergent nucleotide compositions in P. bryantii reporter assay. We show that base composition profoundly affects the amount of the reporter synthesized, and further that these amounts were in agreement with S1 protein binding affinity for adenine/uracil bases in mRNA. This is the first, though indirect, clue that S1 is actually involved in translation initiation in Bacteroidetes and adds the second layer of control beside mRNA secondary structure affecting translation initiation in this phylum.

  11. A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution

    PubMed Central

    Garcia-Moreno, Manuel; Sanz, Miguel Angel; Carrasco, Luis

    2016-01-01

    Sindbis virus (SINV) mRNAs contain several motifs that participate in the regulation of their translation. We have discovered a motif at the 3′ untranslated region (UTR) of viral mRNAs, constituted by three repeated sequences, which is involved in the translation of both SINV genomic and subgenomic mRNAs in insect, but not in mammalian cells. These data illustrate for the first time that an element present at the 3′-UTR confers translatability to mRNAs from an animal virus in a cell-specific manner. Sequences located at the beginning of the 5′-UTR may also regulate SINV subgenomic mRNA translation in both cell lines in a context of infection. Moreover, a replicon derived from Sleeping disease virus, an alphavirus that have no known arthropod vector for transmission, is much more efficient in insect cells when the repeated sequences from SINV are inserted at its 3′-UTR, due to the enhanced translatability of its mRNAs. Thus, these findings provide a clue to understand, at the molecular level, the evolution of alphaviruses and their host range. PMID:26755446

  12. De novo computational identification of stress-related sequence motifs and microRNA target sites in untranslated regions of a plant translatome

    PubMed Central

    Munusamy, Prabhakaran; Zolotarov, Yevgen; Meteignier, Louis-Valentin; Moffett, Peter; Strömvik, Martina V.

    2017-01-01

    Gene regulation at the transcriptional and translational level leads to diversity in phenotypes and function in organisms. Regulatory DNA or RNA sequence motifs adjacent to the gene coding sequence act as binding sites for proteins that in turn enable or disable expression of the gene. Whereas the known DNA and RNA binding proteins range in the thousands, only a few motifs have been examined. In this study, we have predicted putative regulatory motifs in groups of untranslated regions from genes regulated at the translational level in Arabidopsis thaliana under normal and stressed conditions. The test group of sequences was divided into random subgroups and subjected to three de novo motif finding algorithms (Seeder, Weeder and MEME). In addition to identifying sequence motifs, using an in silico tool we have predicted microRNA target sites in the 3′ UTRs of the translationally regulated genes, as well as identified upstream open reading frames located in the 5′ UTRs. Our bioinformatics strategy and the knowledge generated contribute to understanding gene regulation during stress, and can be applied to disease and stress resistant plant development. PMID:28276452

  13. The Effect of Poly(ADP-ribose) Polymerase-1 Gene 3′Untranslated Region Polymorphism in Colorectal Cancer Risk among Saudi Cohort

    PubMed Central

    Alhadheq, Abdullah M.; Purusottapatnam Shaik, Jilani; Alamri, Abdullah; Aljebreen, Abdulrahman M.; Alharbi, Othman; Almadi, Majid A.; Alhadeq, Faten; Azzam, Nahla A.; Alanazi, Mohammad; Bazzi, Mohammad D.

    2016-01-01

    Background. DNA repair systems are essential for each cell to repair and maintain the genome integrity. Base excision repair pathway is one of the crucial pathways to maintain genome integrity and PARP-1 plays a key role in BER pathway. The purpose of this study is to evaluate the association between polymorphisms in PARP-1 3′untranslated region (3′UTR) SNP rs8679 and its expression in colorectal cancer. Methods. Genotyping and gene expression were performed using TaqMan assays. The effects of age, gender, and tumor location were evaluated in cases and controls regarding the genotyping results. Resulting data was analyzed using SPSS software. Results and Conclusions. Genotyping analysis for SNP rs8679 showed decreased susceptibility to colorectal cancer at heterozygous TC allele and at minor allele C. Further this protective association was also observed in younger age patients (≤57), in female patients, and also in patients with tumors located at colon and rectum. PARP-1 expression levels are significantly different in colorectal cancer compared to matched normal tissue. Our findings proved that the upregulation of PARP-1 is associated with tumor progression and poor prognosis in Saudi patients with colorectal cancer, suggesting that PARP-1 can be novel and valuable signatures for predicting the clinical outcome of patients with colorectal cancer. PMID:27746584

  14. Internal initiation of translation directed by the 5'-untranslated region of the tobamovirus subgenomic RNA I(2).

    PubMed

    Skulachev, M V; Ivanov, P A; Karpova, O V; Korpela, T; Rodionova, N P; Dorokhov, Y L; Atabekov, J G

    1999-10-10

    Previously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRES(CP,148)(CR)). The equivalent 148-nt sequence from TMV U1 RNA (U1(CP,148)(SP)) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRES(MP,228)(CR)) contained an IRES element that directed in vitro translation of the 3'-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcripts as well. Consequently this sequence was termed IRES(MP,228)(U1). It was shown that IRES(MP,228)(CR), IRES(MP,228)(U1), and IRES(CP,148)(CR) could mediate expression of the 3'-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRES(MP,75)), which corresponded closely to the length of the 5'UTR of TMV subgenomic RNA (sgRNA) I(2). The RNA transcripts structurally equivalent to I(2) sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRES(MP,75) (HIRES(MP), (75)(CR)-MP-CP-3'UTR; HIRES(MP,75)(U1)-MP-CP-3'UTR), were able to express the MP gene in vitro. The capacity of HIRES(MP,75)(CR) sequence for mediating internal translation of the 3'-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I(2) sgRNAs might proceed via internal ribosome entry pathway mediated by IRES(MP) element contained in the 75-nt 5'UTR

  15. Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-Seq and ESTs.

    PubMed

    Schurch, Nicholas J; Cole, Christian; Sherstnev, Alexander; Song, Junfang; Duc, Céline; Storey, Kate G; McLean, W H Irwin; Brown, Sara J; Simpson, Gordon G; Barton, Geoffrey J

    2014-01-01

    The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3' untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3' polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3' UTR re-annotation (including extension of one 3' UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.

  16. Genome-wide association identifies a deletion in the 3’ untranslated region of Striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy

    PubMed Central

    Meurs, Kathryn M.; Mauceli, Evan; Lahmers, Sunshine; Acland, Gregory M.; White, Stephen N.; Lindblad-Toh, Kerstin

    2010-01-01

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins. We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine-mapping and direct DNA sequencing identified an eight base pair deletion in the 3’ untranslated region (UTR) of the striatin (STRN) gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3’ UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, plakophilin- 2, plakoglobin and desmoplakin, all involved in the pathogenesis of ARVC in human beings. We suggest that striatin may serve as a novel candidate gene for human ARVC. PMID:20596727

  17. Role of the 3'-untranslated region of human endothelin-1 in vascular endothelial cells. Contribution to transcript lability and the cellular heat shock response.

    PubMed

    Mawji, Imtiaz A; Robb, G Brett; Tai, Sharon C; Marsden, Philip A

    2004-03-05

    Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post-transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-1 and luciferase transcripts with or without a wild type ET-1 3'-untranslated region (3'-UTR) and found that the 3'-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10-nucleotide region at position 978-987 of the 3'-UTR. Depletion of AUF1 by RNA interference up-regulated ET-1 in endothelial cells suggesting AUF1-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Taken together, these findings suggest a complex model of ARE-mediated mRNA turnover dependent on two 3'-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock.

  18. A high affinity HSF-1 binding site in the 5'-untranslated region of the murine tumor necrosis factor-alpha gene is a transcriptional repressor.

    PubMed

    Singh, Ishwar S; He, Ju-Ren; Calderwood, Stuart; Hasday, Jeffrey D

    2002-02-15

    Tumor necrosis factor-alpha (TNFalpha) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40 degrees C) markedly attenuates TNFalpha expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFalpha gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5'-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5'-UTR of the TNFalpha gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFalpha gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFalpha promoter (-85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (-1080/+138) TNFalpha promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions -1080/-845, -533/-196, and -326/-39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal -85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNFalpha promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5 degrees C. This suggests that HSF-1 might repress TNFalpha transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.

  19. Tristetraprolin Regulates Interleukin-6 Expression Through p38 MAPK-Dependent Affinity Changes with mRNA 3′ Untranslated Region

    PubMed Central

    Zhao, Wenpu; Liu, Min; D'Silva, Nisha J.

    2011-01-01

    Tristetraprolin (TTP) is a well-characterized, zinc finger-containing, RNA-binding protein. TTP targets tumor necrosis factor α for degradation via the 3′ untranslated region (3′UTR). Although AU-rich elements (AREs) in the 3′UTR of interleukin-6 (IL-6) mRNA dictate mRNA degradation, the role of TTP in the post-transcriptional regulation of IL-6 gene expression is unclear. Here we used TTP-deficient mice to test the hypothesis that IL-6 expression is influenced by TTP. Genetic and siRNA-mediated knockdown of TTP resulted in increased IL-6 production and overexpression of TTP had the reverse effect. IL-6 and tumor necrosis factor α production were elevated after injection of IL-1β in TTP-deficient mice. Further, embryonic fibroblasts from these mice (mouse embryonic fibroblasts) exhibited greater IL-6 mRNA expression and longer half-life than wild-type mouse embryonic fibroblasts. Overexpression of TTP reduced IL-6 3′UTR luciferase reporter activity in an ARE-dependent manner. Proximal and distal regions of the 3′UTR acted synergistically to produce the full repression of TTP. Mutation-based luciferase assays show that ARE2, ARE3, and ARE4 are required for TTP-mediated repression. The constitutively activated p38-MK2 pathway abrogated TTP-mediated repression of IL-6 3′UTR reporter activity. RNA immunoprecipitation assay indicated that the deficiency of p38α resulted in the increased affinity of TTP to IL-6 mRNA. Taken together, we propose that TTP downregulates IL-6 gene expression at the post-transcriptional level by targeting ARE elements in the 3′UTR region. PMID:21457063

  20. The 3'-untranslated region length and AU-rich RNA location modulate RNA-protein interaction and translational control of β2-adrenergic receptor mRNA.

    PubMed

    Subramaniam, Kothandharaman; Kandasamy, Karthikeyan; Joseph, Kusumam; Spicer, Eleanor K; Tholanikunnel, Baby G

    2011-06-01

    Posttranscriptional controls play a major role in β(2)-adrenergic receptor (β(2)-AR) expression. We recently reported that β(2)-AR mRNA translation is suppressed by elements in its 3'-untranslated region (UTR). We also identified T-cell-restricted intracellular antigen-related protein (TIAR) and HuR as prominent AU-rich (ARE) RNA-binding proteins that associate with β(2)-AR mRNA 3'-UTR. In this study, we identified a poly(U) region at the distal end of the 3'-UTR as critical for TIAR binding to β(2)-AR mRNA and for translational suppression. Here, we also report that the locations of the poly(U) and ARE sequences within the 3'-UTR are important determinants that control the translation of β(2)-AR mRNA. Consistent with this finding, a 20-nucleotide ARE RNA from the proximal 3'-UTR that did not inhibit mRNA translation in its native position was able to suppress translation when re-located to the distal 3'-UTR of the receptor mRNA. Immunoprecipitation and polysome profile analysis demonstrated the importance of 3'-UTR length and the ARE RNA location within the 3'-UTR, as key determinants of RNA/protein interactions and translational control of β(2)-AR mRNA. Further, the importance of 3'-UTR length and ARE location in TIAR and HuR association with mRNA and translational suppression was demonstrated using a chimeric luciferase reporter gene.

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

    SciTech Connect

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

    1988-03-01

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

  2. Molecular Mechanism for Hypertensive Renal Disease: Differential Regulation of Chromogranin A Expression at 3'-Untranslated Region Polymorphism C+87T by MicroRNA-107.

    PubMed

    Zhang, Kuixing; Mir, Saiful A; Hightower, C Makena; Miramontes-Gonzalez, Jose Pablo; Maihofer, Adam X; Chen, Yuqing; Mahata, Sushil K; Nievergelt, Caroline M; Schork, Nicholas J; Freedman, Barry I; Vaingankar, Sucheta M; O'Connor, Daniel T

    2015-08-01

    Chromogranin A (CHGA) is coreleased with catecholamines from secretory vesicles in adrenal medulla and sympathetic axons. Genetic variation in the CHGA 3'-region has been associated with autonomic control of circulation, hypertension, and hypertensive nephropathy, and the CHGA 3'-untranslated region (3'-UTR) variant C+87T (rs7610) displayed peak associations with these traits in humans. Here, we explored the molecular mechanisms underlying these associations. C+87T occurred in a microRNA-107 (miR-107) motif (match: T>C), and CHGA mRNA expression varied inversely with miR-107 abundance. In cells transfected with chimeric luciferase/CHGA 3'-UTR reporters encoding either the T allele or the C allele, changes in miR-107 expression levels had much greater effects on expression of the T allele. Cotransfection experiments with hsa-miR-107 oligonucleotides and eukaryotic CHGA plasmids produced similar results. Notably, an in vitro CHGA transcription/translation experiment revealed that changes in hsa-miR-107 expression altered expression of the T allele variant only. Mice with targeted ablation of Chga exhibited greater eGFR. Using BAC transgenesis, we created a mouse model with a humanized CHGA locus (T/T genotype at C+87T), in which treatment with a hsa-miR-107 inhibitor yielded prolonged falls in SBP/DBP compared with wild-type mice. We conclude that the CHGA 3'-UTR C+87T disrupts an miR-107 motif, with differential effects on CHGA expression, and that a cis:trans (mRNA:miR) interaction regulates the association of CHGA with BP and hypertensive nephropathy. These results indicate new strategies for probing autonomic circulatory control and ultimately, susceptibility to hypertensive renal sequelae.

  3. Evolution and Emergence of Enteroviruses through Intra- and Inter-species Recombination: Plasticity and Phenotypic Impact of Modular Genetic Exchanges in the 5' Untranslated Region.

    PubMed

    Muslin, Claire; Joffret, Marie-Line; Pelletier, Isabelle; Blondel, Bruno; Delpeyroux, Francis

    2015-01-01

    Genetic recombination shapes the diversity of RNA viruses, including enteroviruses (EVs), which frequently have mosaic genomes. Pathogenic circulating vaccine-derived poliovirus (cVDPV) genomes consist of mutated vaccine poliovirus (PV) sequences encoding capsid proteins, and sequences encoding nonstructural proteins derived from other species' C EVs, including certain coxsackieviruses A (CV-A) in particular. Many cVDPV genomes also have an exogenous 5' untranslated region (5' UTR). This region is involved in virulence and includes the cloverleaf (CL) and the internal ribosomal entry site, which play major roles in replication and the initiation of translation, respectively. We investigated the plasticity of the PV genome in terms of recombination in the 5' UTR, by developing an experimental model involving the rescue of a bipartite PV/CV-A cVDPV genome rendered defective by mutations in the CL, following the co-transfection of cells with 5' UTR RNAs from each of the four human EV species (EV-A to -D). The defective cVDPV was rescued by recombination with 5' UTR sequences from the four EV species. Homologous and nonhomologous recombinants with large deletions or insertions in three hotspots were isolated, revealing a striking plasticity of the 5' UTR. By contrast to the recombination of the cVDPV with the 5' UTR of group II (EV-A and -B), which can decrease viral replication and virulence, recombination with the 5' UTRs of group I (EV-C and -D) appeared to be evolutionarily neutral or associated with a gain in fitness. This study illustrates how the genomes of positive-strand RNA viruses can evolve into mosaic recombinant genomes through intra- or inter-species modular genetic exchanges, favoring the emergence of new recombinant lineages.

  4. Evolution and Emergence of Enteroviruses through Intra- and Inter-species Recombination: Plasticity and Phenotypic Impact of Modular Genetic Exchanges in the 5’ Untranslated Region

    PubMed Central

    Muslin, Claire; Joffret, Marie-Line; Pelletier, Isabelle; Blondel, Bruno; Delpeyroux, Francis

    2015-01-01

    Genetic recombination shapes the diversity of RNA viruses, including enteroviruses (EVs), which frequently have mosaic genomes. Pathogenic circulating vaccine-derived poliovirus (cVDPV) genomes consist of mutated vaccine poliovirus (PV) sequences encoding capsid proteins, and sequences encoding nonstructural proteins derived from other species’ C EVs, including certain coxsackieviruses A (CV-A) in particular. Many cVDPV genomes also have an exogenous 5’ untranslated region (5’ UTR). This region is involved in virulence and includes the cloverleaf (CL) and the internal ribosomal entry site, which play major roles in replication and the initiation of translation, respectively. We investigated the plasticity of the PV genome in terms of recombination in the 5’ UTR, by developing an experimental model involving the rescue of a bipartite PV/CV-A cVDPV genome rendered defective by mutations in the CL, following the co-transfection of cells with 5’ UTR RNAs from each of the four human EV species (EV-A to -D). The defective cVDPV was rescued by recombination with 5’ UTR sequences from the four EV species. Homologous and nonhomologous recombinants with large deletions or insertions in three hotspots were isolated, revealing a striking plasticity of the 5’ UTR. By contrast to the recombination of the cVDPV with the 5’ UTR of group II (EV-A and -B), which can decrease viral replication and virulence, recombination with the 5’ UTRs of group I (EV-C and -D) appeared to be evolutionarily neutral or associated with a gain in fitness. This study illustrates how the genomes of positive-strand RNA viruses can evolve into mosaic recombinant genomes through intra- or inter-species modular genetic exchanges, favoring the emergence of new recombinant lineages. PMID:26562151

  5. A 205-Nucleotide Deletion in the 3′ Untranslated Region of Avian Leukosis Virus Subgroup J, Currently Emergent in China, Contributes to Its Pathogenicity

    PubMed Central

    Wang, Qi; Gao, Yulong; Wang, Yongqiang; Qin, Liting; Qi, Xiaole; Qu, Yue; Gao, Honglei

    2012-01-01

    In the past 5 years, an atypical clinical outbreak of avian leukosis virus subgroup J (ALV-J), which contains a unique 205-nucleotide deletion in its 3′ untranslated region (3′UTR), has become epidemic in chickens in China. To determine the role of the 205-nucleotide deletion in the pathogenicity of ALV-J, a pair of viruses were constructed and rescued. The first virus was an ALV-J Chinese isolate (designated HLJ09SH01) containing the 205-nucleotide deletion in its 3′UTR. The second virus was a chimeric clone in which the 3′UTR contains a 205-nucleotide sequence corresponding to a region of the ALV-J prototype virus. The replication and pathogenicity of the rescued viruses (rHLJ09SH01 and rHLJ09SH01A205) were investigated. Compared to rHLJ09SH01A205, rHLJ09SH01 showed a moderate growth advantage in vitro and in vivo, in addition to exhibiting a higher oncogenicity rate and lethality rate in layers and broilers. Increased vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth receptor subtype 2 (VEGFR-2) expression was induced by rHLJ09SH01 more so than by rHLJ09SH01A205 during early embryonic vascular development, but this increased expression disappeared when the expression levels were normalized to the viral levels. This finding suggests that the expression of VEGF-A and VEGFR-2 is associated with viral replication and may also represent a novel molecular mechanism underlying the oncogenic potential of ALV-J. Overall, our findings not only indicate that the unique 205-nucleotide deletion in the ALV-J genome occurred naturally in China and contributes to increased pathogenicity but also point to the possible mechanism of ALV-J-induced oncogenicity. PMID:22993155

  6. Identification and sexually dimorphic expression of vasa isoforms in Dabry's sturgeon (Acipenser dabryanus), and functional analysis of vasa 3'-untranslated region.

    PubMed

    Ye, Huan; Yue, Hua-Mei; Yang, Xiao-Ge; Li, Chuang-Ju; Wei, Qi-Wei

    2016-10-01

    Germ cells are set aside from somatic cells early in embryogenesis, and are responsible for transmitting genetic information through generations. Vasa is a highly conserved germ cell marker across animal phyla, and widely used to label primordial germ cells. Dabry's sturgeon is a rare and endangered species distributed solely in the Yangtze River basin. Here, seven vasa isoforms, named Advasa1-7, were isolated and characterized in Dabry's sturgeon. RT-PCR and western blot analyses revealed that vasa mRNA and protein were mainly restricted to the testis and ovary, but exhibited sexually dimorphic expression. Cellular and subcellular localization uncovered that Advasa mRNA and protein displayed mitotic and meiotic expression in females, and mainly showed mitotic expression in males; surprisingly, they exhibited both cytoplasmic and nuclear expression in the ovarian germ cells, while showing exclusively cytoplasmic expression in the testicular germ cells. By microinjecting chimeric RNA consisting of the red fluorescent protein coding region and the Advasa 3'-untranslated region into embryos of Dabry's sturgeon, zebrafish and medaka, we demonstrated that it had the ability to visualize primordial germ cells (PGCs) in Dabry's sturgeon and zebrafish but not in medaka. It seemed that the machinery of vasa 3'UTR RNA localization was conserved between Dabry's sturgeon and ostariophysan, while possibly changed during the divergence of euteleosts and ostariophysan. Finally, Dabry's sturgeon PGCs moved on the yolk ball, and migrated toward the genital ridge via mesenchyme. Taken together, these results provide new information for vasa expression pattern and function, and lay a foundation for PGC cryopreservation and conservation of Dabry's sturgeon.

  7. Characterisation of genotypes among bovine viral diarrhoea virus 2 strains according to palindromic nucleotide substitutions in the 5 untranslated genomic region.

    PubMed

    Giangaspero, M; Harasawa, R

    2004-01-01

    Pestivirus bovine viral diarrhea virus 2 (BVDV-2) strains from 61 isolates from cattle and sheep, and from some adventitious contaminants of biologicals, have been assessed using the palindromic nucleotide substitutions (PNS) method at three variable loci (V1, V2 and V3) located delin the 5' untranslated region (UTR) of genomic RNA. This genotyping procedure is new, simple and practical. Two characteristics of the base pairings common to BVDV-2 species, a C-G or U-A pairing at the V1 locus, and a G*U pairing at the V2 locus, were observed in isolates tested. The PNS method showed six genotypes: BVDV-2a, BVDV-2b, BVDV-2c, BVDV- 2d, BVDV-2e and BVDV-2f. Twenty-five strains showed the BVDV-2a genotype specific combination of three base pairings (A-U in position 1 and C-G or U*G in position 18 in V1 and U-A or U*G in position 4 in V2). Ten strains were identified by a single C-G pairing in position 4 from the bottom of the V2 stem region, characteristic to genotype BVDV-2b. Three strains were assigned to genotype BVDV-2c, due to their recognition by a G*U pairing at the bottom of the V1 stem region. A U-A pairing, characteristic of the BVDV-2d genotype when found in position 18 of the V1 stem region, was observed in fourteen strains. Genotype BVDV- 2e, present in only six South American cattle isolates, was characterized by G-C pairing in position 12, by U-A pairing in position 16 and G_G or G-_A bulges in position 18 in the V1 region. One strain from Argentina was classified as genotype BVDV-2f, showing: A-U pairing in position 9 and 12, U-A in position 16 and G_A bulge in position 18 in V1 region. Two strains were not characterized due to incomplete sequence of V1 locus.

  8. Comparative Complete Genome Analysis of Chicken and Turkey Megriviruses (Family Picornaviridae): Long 3′ Untranslated Regions with a Potential Second Open Reading Frame and Evidence for Possible Recombination

    PubMed Central

    Boros, Ákos; Pankovics, Péter; Knowles, Nick J.; Nemes, Csaba; Delwart, Eric

    2014-01-01

    ABSTRACT Members of the family Picornaviridae consist of small positive-sense single-stranded RNA (+ssRNA) viruses capable of infecting various vertebrate species, including birds. One of the recently identified avian picornaviruses, with a remarkably long (>9,040-nucleotide) but still incompletely sequenced genome, is turkey hepatitis virus 1 (THV-1; species Melegrivirus A, genus Megrivirus), a virus associated with liver necrosis and enteritis in commercial turkeys (Meleagris gallopavo). This report presents the results of the genetic analysis of three complete genomes of megriviruses from fecal samples of chickens (chicken/B21-CHV/2012/HUN, GenBank accession no. KF961186, and chicken/CHK-IV-CHV/2013/HUN, GenBank accession no. KF961187) (Gallus gallus domesticus) and turkey (turkey/B407-THV/2011/HUN, GenBank accession no. KF961188) (Meleagris gallopavo) with the largest picornavirus genome (up to 9,739 nucleotides) so far described. The close phylogenetic relationship to THV-1 in the nonstructural protein-coding genome region and possession of the same internal ribosomal entry site type (IVB-like) suggest that the study strains belong to the genus Megrivirus. However, the genome comparisons revealed numerous unique variations (e.g., different numbers of potential 2A peptides, unusually long 3′ genome parts with various lengths of a potential second open reading frame, and multiple repeating sequence motifs in the 3′ untranslated region) and heterogeneous sequence relationships between the structural and nonstructural genome regions. These differences suggest the classification of chicken megrivirus-like viruses into a candidate novel species in the genus Megrivirus. Based on the different phylogenetic positions of chicken megrivirus-like viruses at the structural and nonstructural genome regions, the recombinant nature of these viruses is plausible. IMPORTANCE The comparative genome analysis of turkey and novel chicken megriviruses revealed numerous unique

  9. G-Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress.

    PubMed

    Lee, Sang C; Zhang, Jack; Strom, Josh; Yang, Danzhou; Dinh, Thai Nho; Kappeler, Kyle; Chen, Qin M

    2017-01-01

    Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did posttranslational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress. Copyright © 2016 American Society for Microbiology.

  10. Polypyrimidine tract-binding protein binds to the 5' untranslated region of the mouse mammary tumor virus mRNA and stimulates cap-independent translation initiation.

    PubMed

    Cáceres, Carlos J; Contreras, Nataly; Angulo, Jenniffer; Vera-Otarola, Jorge; Pino-Ajenjo, Constanza; Llorian, Miriam; Ameur, Melissa; Lisboa, Francisco; Pino, Karla; Lowy, Fernando; Sargueil, Bruno; López-Lastra, Marcelo

    2016-05-01

    The 5' untranslated region (UTR) of the full-length mRNA of the mouse mammary tumor virus (MMTV) harbors an internal ribosomal entry site (IRES). In this study, we show that the polypyrimidine tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the MMTV 5' UTR stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Results show that PTB1 and PTB4, but not PTB2, stimulate MMTV-IRES activity. PTB1 promotes MMTV-IRES-mediated initiation more strongly than PTB4. When expressed in combination, PTB1 further enhanced PTB4 stimulation of the MMTV-IRES, while PTB2 fully abrogates PTB4-induced stimulation. PTB1-induced stimulation of MMTV-IRES was not altered in the presence of PTB4 or PTB2. Mutational analysis reveals that stimulation of MMTV-IRES activity is abrogated when PTB1 is mutated either in RRM1/RRM2 or RRM3/RRM4. In contrast, a PTB4 RRM1/RRM2 mutant has reduced effect over MMTV-IRES activity, while stimulation of the MMTV-IRES activity is still observed when the PTB4 RRM3/RMM4 mutant is used. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity. In contrast, PTB2 acts as a negative modulator of PTB4-induced stimulation of MMTV-IRES. We conclude that PTB1 and PTB4 act as IRES trans-acting factors of the MMTV-IRES.

  11. Association of a 3' Untranslated Region Polymorphism in PCSK9 with HIV Viral Load and CD4+ Levels in HIV/Hepatitis C Virus Co-Infected Women.

    PubMed

    Kuniholm, Mark H; Liang, Hua; Anastos, Kathryn; Gustafson, Deborah; Kassaye, Seble; Nowicki, Marek; Sha, Beverly E; Pawlowski, Emilia J; Gange, Stephen J; Aouizerat, Bradley E; Pushkarsky, Tatiana; Bukrinsky, Michael I; Prasad, Vinayaka R

    2017-09-18

    To assess variation in genes that regulate cholesterol metabolism in relation to the natural history of HIV infection. Cross-sectional and longitudinal analysis of the Women's Interagency HIV Study (WIHS). We examined 2,050 single nucleotide polymorphisms (SNPs) in 19 genes known to regulate cholesterol metabolism in relation to HIV viral load and CD4 T cell levels in a multiracial cohort of 1,066 antiretroviral therapy (ART) naïve women. Six SNPs were associated with both HIV viral load and CD4 T cell levels at a false discovery rate (FDR) of 0.01. Bioinformatics tools did not predict functional activity for five SNPs, located in introns of NCOR2, RXRA and TTC39B. Rs17111557 located in the 3' untranslated region (UTR) of PCSK9 putatively affects binding of hsa-miR-548t-5p and hsa-miR-4796-3p, which could regulate PCSK9 expression levels. Interrogation of rs17111557 revealed stronger associations in the subset of women with HIV/hepatitis C virus (HCV) co-infection (n = 408, 38% of women). Rs17111557 was also associated with low-density lipoprotein cholesterol (LDL-C) levels in HIV/HCV co-infected (β: -10.4; 95% CI: -17.9, -2.9; P = 0.007), but not in HIV monoinfected (β:1.2; 95% CI: -6.3, 8.6; P = 0.76) women in adjusted analysis. PCSK9 polymorphism may affect HIV pathogenesis, particularly in HIV/HCV co-infected women. A likely mechanism for this effect is PCSK9-mediated regulation of cholesterol metabolism. Replication in independent cohorts is needed to clarify the generalizability of the observed associations.

  12. Mini-genome rescue of Crimean-Congo hemorrhagic fever virus and research into the evolutionary patterns of its untranslated regions.

    PubMed

    Zhao, Jiuru; Xia, Han; Zhang, Yujiang; Yin, Shiyu; Zhang, Zhong; Tang, Shuang; Kou, Zheng; Yu, Jingfeng; Fan, Zhaojun; Li, Tianxian

    2013-10-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of genus Nairovirus, family Bunyaviridae, which are distributed widely in Africa, Europe and Asia with several genotypes. As a BSL-4 level pathogen, the requirement of high-level biosafety facilities severely constrains researches on live virus manipulation. In this study, we developed a helper-virus-independent mini-genome rescue system for the Chinese YL04057 strain. Based on the enhanced green fluorescent protein (EGFP)-derived mini-genome plasmids, this polymerase I driven system permits easy observation and quantification. Unlike previous report, gradually reduced levels of activity of the CCHFV L, M and S untranslated regions (UTRs) were observed in our system. We also demonstrated that the UTRs at both ends were indispensable for mini-genome background expression. In addition, we phylogentically analyzed all six UTRs of CCHFV and showed that L-UTRs were clustered together approximately corresponding to their original geographical continents. The UTRs of M segment showed a similar branch structure to its open reading frames (ORFs), and nearly an identical tree was generated with 5' UTRs of S segment compared with its ORFs. However, the 3' UTRs of S segment formed new divergent groups. Compatibility tests of YL04057 strain nucleocapsid protein and L protein expression plasmids with Nigerian strain IbAr10200 mini-genomes revealed lower compatibility of L-UTRs without an obvious effect on M-UTRs. Moreover, we demonstrated that the L-UTRs could tolerate certain nucleotide mutations. This system may provide a foundation for future studies of the viral replication cycle, pathogenic mechanisms and evolutionary patterns of CCHFV.

  13. Spliceosomal introns in the 5' untranslated region of plant BTL RING-H2 ubiquitin ligases are evolutionary conserved and required for gene expression.

    PubMed

    Aguilar-Hernández, Victor; Guzmán, Plinio

    2013-11-14

    Introns located close to the 5' end of a gene or in the 5' untranslated region often exert positive effects on gene expression. This effect, known as intron-mediated enhancement (IME), has been observed in diverse eukaryotic organisms, including plants. The sequences involved in IME seem to be spread across the intron and function in an additive manner. The IMEter algorithm was developed to predict plant introns that may enhance gene expression. We have identified several plant members of the BTL class of E3s, which may have orthologs across eukaryotes, that contain a 5'UTR intron. The RING finger E3 ligases are key enzymes of the ubiquitination system that mediate the transfer of ubiquitin to substrates. In this study, we retrieved BTL sequences from several angiosperm species and found that 5'UTR introns showing a strong IMEter score were predicted, suggesting that they may be conserved by lineage. Promoter-GUS fusion lines were used to confirm the IME effect of these 5'UTR introns on gene expression. IMEter scores of BTLs were compared with the 5'UTR introns of two gene families MHX and polyubiquitin genes. Analysis performed in two Arabidopsis BTL E3 ligases genes indicated that the 5'UTR introns were essential for gene expression in all the tissues tested. Comparison of the average 5'UTR intron size on three gene families in ten angiosperm species suggests that a prevalent size for a 5'UTR intron is in the range of 600 nucleotides, and that the overall IMEter score within a gene family is preserved across several angiosperms. Our results indicated that gene expression dependent on a 5'UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution.

  14. The 811 C/T polymorphism in the 3' untranslated region of the selenoprotein 15-kDa (Sep15) gene and breast cancer in Caucasian women.

    PubMed

    Watrowski, Rafał; Castillo-Tong, Dan Cacsire; Fabjani, Gerhild; Schuster, Eva; Fischer, Michael; Zeillinger, Robert

    2016-01-01

    The 15-kDa selenoprotein (Sep15) is a selenocysteine-containing oxidoreductase in the endoplasmic reticulum that participates in disulfide-bond formation and protein folding control. The 3'-untranslated region (3'-UTR) contains two exclusively linked, polymorphic sites at positions 811 (C/T) and 1125 (G/A), which result in two functional haplotypes: 811C/1125G or 811T/1125A. The 811T/1125A variant occurs significantly more often in African-Americans as compared to Caucasians and has been linked to increased breast cancer risk in black women. We studied the 811C/T (rs5845) Sep15 gene polymorphism in 182 Caucasian women-83 breast cancer cases and 99 healthy controls-by pyrosequencing and polymerase chain reaction. Associations between allelic variants and clinico-pathological variables (e.g., age, stage of disease, tumor type, grading, and receptor status) were investigated. The genotype distribution in breast cancer patients (CC 63.9 %, CT 33.7 %, TT 2.4 %) and controls (69.7 %, CT 28.3 %, TT 2 %) showed no significant difference (OR 0.77, 95 % CI 0.41-1.42, p = 0.4). The overall low prevalence of the T allele was in accordance with that reported for Caucasians in previous studies. There was no significant association between 811C/T Sep15 polymorphism and any of clinico-pathological parameters. In conclusion, we are the first to report on 811C/T SEP 15 polymorphism in white breast cancer patients. Genotype variation within the 3'-UTR of the SEP 15 gene showed no association with breast cancer risk or clinico-pathological parameters in Caucasian women.

  15. Regulation of ATF1 and ATF2 transcripts by sequences in their 3' untranslated region in cleavage-stage cattle embryos.

    PubMed

    Orozco-Lucero, Ernesto; Dufort, Isabelle; Sirard, Marc-André

    2017-04-01

    The sequence of a 3' untranslated region (3'UTR) of mRNA governs the timing of its polyadenylation and translation in mammalian oocytes and early embryos. The objective of this study was to assess the influence of cis-elements in the 3'UTR of the developmentally important ATF1 and ATF2 transcripts on their timely translation during first cleavages in bovine embryos. Eight different reporter mRNAs (coding sequence of green fluorescent protein [GFP] fused to the 3'UTR of short or long isoforms of cattle ATF1 or -2, with or without polyadenylation) or a control GFP mRNA were microinjected separately into presumptive bovine zygotes at 18 hr post-insemination (hpi), followed by epifluorescence assessment for GFP translation between 24 and 80 hpi (expressed as percentage of GFP-positive embryos calculated from the total number of individuals). The presence of either polyadenine or 3'UTR sequence in deadenylated constructs is required for GFP translation (implying the need for polyadenylation), and all exogenous mRNAs that met either criteria were translated as soon as 24 hpi-except for long-deadenylated ATF2-UTR, whose translation began at 36 hpi. Overall, GFP was more visibly translated in competent (cleaving) embryos, particularly in long ATF1/2 constructs. The current data shows a timely GFP translation in bovine embryos depending on sequences in the 3'UTR of ATF1/2, and indicates a difference between short and long isoforms. In addition, cleaving embryos displayed increased translational capacity of the tested constructs. Functional confirmation of the identification cis-sequences in the 3'UTR of ATF1/2 will contribute to the understanding of maternal mRNA translation regulation during early cattle development. © 2017 Wiley Periodicals, Inc.

  16. Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3'-untranslated region of their mRNAs.

    PubMed

    Roca, Francisco J; Cayuela, María L; Secombes, Chris J; Meseguer, José; Mulero, Victoriano

    2007-01-01

    The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.

  17. A variable number of tandem repeats in the 3'-untranslated region of the dopamine transporter modulates striatal function during working memory updating across the adult age span.

    PubMed

    Sambataro, Fabio; Podell, Jamie E; Murty, Vishnu P; Das, Saumitra; Kolachana, Bhaskar; Goldberg, Terry E; Weinberger, Daniel R; Mattay, Venkata S

    2015-08-01

    Dopamine modulation of striatal function is critical for executive functions such as working memory (WM) updating. The dopamine transporter (DAT) regulates striatal dopamine signaling via synaptic reuptake. A variable number of tandem repeats in the 3'-untranslated region of SLC6A3 (DAT1-3'-UTR-VNTR) is associated with DAT expression, such that 9-repeat allele carriers tend to express lower levels (associated with higher extracellular dopamine concentrations) than 10-repeat homozygotes. Aging is also associated with decline of the dopamine system. The goal of the present study was to investigate the effects of aging and DAT1-3'-UTR-VNTR on the neural activity and functional connectivity of the striatum during WM updating. Our results showed both an age-related decrease in striatal activity and an effect of DAT1-3'-UTR-VNTR. Ten-repeat homozygotes showed reduced striatal activity and increased striatal-hippocampal connectivity during WM updating relative to the 9-repeat carriers. There was no age by DAT1-3'-UTR-VNTR interaction. These results suggest that, whereas striatal function during WM updating is modulated by both age and genetically determined DAT levels, the rate of the age-related decline in striatal function is similar across both DAT1-3'-UTR-VNTR genotype groups. They further suggest that, because of the baseline difference in striatal function based on DAT1-3'-UTR-VNTR polymorphism, 10-repeat homozygotes, who have lower levels of striatal function throughout the adult life span, may reach a threshold of decreased striatal function and manifest impairments in cognitive processes mediated by the striatum earlier in life than the 9-repeat carriers. Our data suggest that age and DAT1-3'-UTR-VNTR polymorphism independently modulate striatal function.

  18. microRNA-558 facilitates the expression of hypoxia-inducible factor 2 alpha through binding to 5′-untranslated region in neuroblastoma

    PubMed Central

    Song, Huajie; Jiao, Wanju; Li, Dan; Fang, Erhu; Wang, Xiaojing; Mei, Hong; Pu, Jiarui; Huang, Kai; Tong, Qiangsong

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Our previous studies have shown that hypoxia-inducible factor 2 alpha (HIF-2α), one member of the bHLH-PAS transcription factor family, facilitates the progression of NB under non-hypoxic conditions. However, the mechanisms underlying HIF-2α expression in NB still remain largely unknown. Herein, through analyzing the computational algorithm programs, we identified microRNA-558 (miR-558) as a crucial regulator of HIF-2α expression in NB. We demonstrated that miR-558 promoted the expression of HIF-2α at translational levels in NB cells through recruiting Argonaute 2 (AGO2). Mechanistically, miR-558 directly bound with its complementary site within 5′-untranslated region (5′-UTR) to facilitate the binding of AGO2 to eukaryotic translation initiation factor 4E (eIF4E) binding protein 1, resulting in increased eIF4E enrichment and HIF-2α translation. In addition, miR-558 promoted the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo, and these biological features were rescued by knockdown of AGO2, eIF4E, or HIF-2α. In clinical NB specimens, miR-558, AGO2, and eIF4E were highly expressed and positively correlated with HIF-2α expression. Patients with high miR-558, HIF-2α, AGO2, or eIF4E levels had lower survival probability. Taken together, these results demonstrate that miR-558 facilitates the expression of HIF-2α through bindingto its 5′-UTR, thus promoting the tumorigenesis and aggressiveness of NB. PMID:27276678

  19. The 5' untranslated region of the human T-cell lymphotropic virus type 1 mRNA enables cap-independent translation initiation.

    PubMed

    Olivares, Eduardo; Landry, Dori M; Cáceres, C Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R; López-Lastra, Marcelo

    2014-06-01

    The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5'-untranslated region (5'UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5'UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression.

  20. The 3′ untranslated region of a soybean cytosolic glutamine synthetase (GS1) affects transcript stability and protein accumulation in transgenic alfalfa

    PubMed Central

    Ortega, Jose L.; Moguel-Esponda, Salvador; Potenza, Carol; Conklin, Cristina F.; Quintana, Anita; Sengupta-Gopalan, Champa

    2013-01-01

    Summary Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). Glutamine synthetase 1 is regulated in different plants at the transcriptional level and there are some reports of regulation at the level of protein stability. Here we present data that clearly establish that GS1 in plants is also regulated at the level of transcript turnover and at the translational level. Using a Glycine max (soybean) GS1 transgene, with and without its 3′ untranslated region (UTR), driven by the constitutive CaMV 35S promoter in Medicago sativa (alfalfa) and Nicotiana tabacum (tobacco), we show that the 3′ UTR plays a major role in both transcript turnover and translation repression in both the leaves and the nodules. Our data suggest that the 3′ UTR mediated turnover of the transcript is regulated by a nitrogen metabolite or carbon/nitrogen ratios. We also show that the 3′ UTR of the gene for the soybean GS1 confers post-transcriptional regulation on a reporter gene. Our dissection of post-transcriptional and translational levels of regulation of GS in plants shows that the situation in plants strongly resembles that in other organisms where GS is regulated at almost all levels. Multistep regulation of GS shows the high priority given by organisms to regulating and ensuring optimal control of nitrogen substrates and preventing overproduction of glutamine and drainage of the glutamate pool. PMID:16460515

  1. Yeast Edc3 Targets RPS28B mRNA for Decapping by Binding to a 3′ Untranslated Region Decay-Inducing Regulatory Element

    PubMed Central

    Li, Chunfang; Roy, Bijoyita

    2014-01-01

    mRNA decapping commits a transcript to complete turnover in eukaryotic cells. In yeast, general mRNA decapping requires the Dcp1/Dcp2 decapping enzyme and a set of decapping activators, including Pat1, Dhh1, Edc3, and the Lsm1-7 complex. The exact function and mode of action of each of these decapping activators in mRNA decapping largely remain elusive. Here, we analyzed the role of Edc3 in the decay of yeast RPS28B mRNA, a pathway triggered by a negative-feedback autoregulatory mechanism. We show that Edc3-mediated RPS28B mRNA decay requires either of two orthologous proteins, Rps28a and Rps28b, expressed from the RPS28A and RPS28B genes, respectively. Contrary to a generally accepted model, we found that Rps28b does not bind to the 3′-untranslated region (UTR) regulatory element in RPS28B mRNA. Instead, Edc3 is directly involved in binding the element, and Rps28b binds Edc3 and regulates its activity. Decay of RPS28B mRNA requires the Lsm and YjeF-N domains of Edc3, but surprisingly, decay of YRA1 pre-mRNA, the only other known substrate of Edc3, requires only the Lsm domain. Collectively, our experiments reveal a new role for Edc3 in mRNA substrate recognition and suggest that this activity is subject to intricate regulation by additional factors, including the Rps28 ribosomal protein. PMID:24492965

  2. The role of CYP3A4 mRNA transcript with shortened 3'-untranslated region in hepatocyte differentiation, liver development, and response to drug induction.

    PubMed

    Li, Dan; Gaedigk, Roger; Hart, Steven N; Leeder, J Steven; Zhong, Xiao-bo

    2012-01-01

    Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3'-untranslated region (UTR), one had a shorter 3'-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3'-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3'-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3'-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3'-UTR. We conclude that the 3'-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction.

  3. Spliceosomal introns in the 5′ untranslated region of plant BTL RING-H2 ubiquitin ligases are evolutionary conserved and required for gene expression

    PubMed Central

    2013-01-01

    Background Introns located close to the 5′ end of a gene or in the 5′ untranslated region often exert positive effects on gene expression. This effect, known as intron-mediated enhancement (IME), has been observed in diverse eukaryotic organisms, including plants. The sequences involved in IME seem to be spread across the intron and function in an additive manner. The IMEter algorithm was developed to predict plant introns that may enhance gene expression. We have identified several plant members of the BTL class of E3s, which may have orthologs across eukaryotes, that contain a 5′UTR intron. The RING finger E3 ligases are key enzymes of the ubiquitination system that mediate the transfer of ubiquitin to substrates. Results In this study, we retrieved BTL sequences from several angiosperm species and found that 5′UTR introns showing a strong IMEter score were predicted, suggesting that they may be conserved by lineage. Promoter-GUS fusion lines were used to confirm the IME effect of these 5′UTR introns on gene expression. IMEter scores of BTLs were compared with the 5′UTR introns of two gene families MHX and polyubiquitin genes. Conclusions Analysis performed in two Arabidopsis BTL E3 ligases genes indicated that the 5′UTR introns were essential for gene expression in all the tissues tested. Comparison of the average 5′UTR intron size on three gene families in ten angiosperm species suggests that a prevalent size for a 5′UTR intron is in the range of 600 nucleotides, and that the overall IMEter score within a gene family is preserved across several angiosperms. Our results indicated that gene expression dependent on a 5′UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution. PMID:24228887

  4. The (95)(Δ)G mutation in the 5'untranslated region of the norA gene increases efflux activity in Staphylococcus epidermidis isolates.

    PubMed

    García-Gómez, Elizabeth; Jaso-Vera, Marcos E; Juárez-Verdayes, Marco A; Alcántar-Curiel, María D; Zenteno, Juan C; Betanzos-Cabrera, Gabriel; Peralta, Humberto; Rodríguez-Martínez, Sandra; Cancino-Díaz, Mario E; Jan-Roblero, Janet; Cancino-Diaz, Juan C

    2017-02-01

    In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR (95)(Δ)G mutation. Isolates carrying the (95)(Δ)G mutation had higher levels of norA expression compared with those that did not. To corroborate that the (95)(Δ)G mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the (95)(Δ)G mutation and had a high level of norA expression and EF, indicating that the (95)(Δ)G mutation is important for the HEA phenotype. The (95)(Δ)G mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR (95)(Δ)G mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.

  5. Effect of β2-adrenergic receptor gene (ADRB2) 3′ untranslated region polymorphisms on inhaled corticosteroid/long-acting β2-adrenergic agonist response

    PubMed Central

    2012-01-01

    Background Evidence suggests that variation in the length of the poly-C repeat in the 3′ untranslated region (3′UTR) of the β2-adrenergic receptor gene (ADRB2) may contribute to interindividual variation in β-agonist response. However, methodology in previous studies limited the assessment of the effect of sequence variation in the context of poly-C repeat length. The objectives of this study were to design a novel genotyping method to fully characterize sequence variation in the ADRB2 3′UTR poly-C repeat in asthma patients treated with inhaled corticosteroid and long-acting β2-adrenergic agonist (ICS/LABA) combination therapy, and to analyze the effect of the poly-C repeat polymorphism on clinical response. Methods In 2,250 asthma patients randomized to treatment with budesonide/formoterol or fluticasone/salmeterol in a six-month study (AstraZeneca study code: SD-039-0735), sequence diversity in the ADRB2 poly-C repeat region was determined using a novel sequencing-based genotyping method. The relationship between the poly-C repeat polymorphism and the incidence of severe asthma exacerbations, and changes in pulmonary function and asthma symptoms from baseline to the average during the treatment period, were analyzed. Results Poly-C repeat genotypes were assigned in 97% (2,192/2,250) of patients. Of the 13 different poly-C repeat alleles identified, six alleles occurred at a frequency of >5% in one or more population in this study. The repeat length of these six common alleles ranged from 10 to 14 nucleotides. Twelve poly-C repeat genotypes were observed at a frequency of >1%. No evidence of an association between poly-C repeat genotype and the incidence of severe asthma exacerbations was observed. Patients’ pulmonary function measurements improved and asthma symptoms declined when treated with ICS/LABA combination therapy regardless of poly-C repeat genotype. Conclusions The extensive sequence diversity present in the poly-C repeat region of the ADRB2

  6. 5[prime] to 3[prime] nucleic acid synthesis using 3[prime]-photoremovable protecting group

    DOEpatents

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

    1999-06-01

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

  7. Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

    NASA Technical Reports Server (NTRS)

    Mazumder, B.; Fox, P. L.

    1999-01-01

    Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine

  8. A partial MECP2 duplication in a mildly affected adult male: a putative role for the 3' untranslated region in the MECP2 duplication phenotype

    PubMed Central

    2012-01-01

    Background Duplications of the X-linked MECP2 gene are associated with moderate to severe intellectual disability, epilepsy, and neuropsychiatric illness in males, while triplications are associated with a more severe phenotype. Most carrier females show complete skewing of X-inactivation in peripheral blood and an apparent susceptibility to specific personality traits or neuropsychiatric symptoms. Methods We describe the clinical phenotype of a pedigree segregating a duplication of MECP2 found on clinical array comparative genomic hybridization. The position, size, and extent of the duplication were delineated in peripheral blood samples from affected individuals using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization, as well as targeted high-resolution oligonucleotide microarray analysis and long-range PCR. The molecular consequences of the rearrangement were studied in lymphoblast cell lines using quantitative real-time PCR, reverse transcriptase PCR, and western blot analysis. Results We observed a partial MECP2 duplication in an adult male with epilepsy and mild neurocognitive impairment who was able to function independently; this phenotype has not previously been reported among males harboring gains in MECP2 copy number. The same duplication was inherited by this individual’s daughter who was also affected with neurocognitive impairment and epilepsy and carried an additional copy-number variant. The duplicated segment involved all four exons of MECP2, but excluded almost the entire 3' untranslated region (UTR), and the genomic rearrangement resulted in a MECP2-TEX28 fusion gene mRNA transcript. Increased expression of MECP2 and the resulting fusion gene were both confirmed; however, western blot analysis of lysates from lymphoblast cells demonstrated increased MeCP2 protein without evidence of a stable fusion gene protein product. Conclusion The observations of a mildly affected adult male with a MECP2 duplication and

  9. A partial MECP2 duplication in a mildly affected adult male: a putative role for the 3' untranslated region in the MECP2 duplication phenotype.

    PubMed

    Hanchard, Neil A; Carvalho, Claudia M B; Bader, Patricia; Thome, Aaron; Omo-Griffith, Lisa; del Gaudio, Daniela; Pehlivan, Davut; Fang, Ping; Schaaf, Christian P; Ramocki, Melissa B; Lupski, James R; Cheung, Sau Wai

    2012-08-10

    Duplications of the X-linked MECP2 gene are associated with moderate to severe intellectual disability, epilepsy, and neuropsychiatric illness in males, while triplications are associated with a more severe phenotype. Most carrier females show complete skewing of X-inactivation in peripheral blood and an apparent susceptibility to specific personality traits or neuropsychiatric symptoms. We describe the clinical phenotype of a pedigree segregating a duplication of MECP2 found on clinical array comparative genomic hybridization. The position, size, and extent of the duplication were delineated in peripheral blood samples from affected individuals using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization, as well as targeted high-resolution oligonucleotide microarray analysis and long-range PCR. The molecular consequences of the rearrangement were studied in lymphoblast cell lines using quantitative real-time PCR, reverse transcriptase PCR, and western blot analysis. We observed a partial MECP2 duplication in an adult male with epilepsy and mild neurocognitive impairment who was able to function independently; this phenotype has not previously been reported among males harboring gains in MECP2 copy number. The same duplication was inherited by this individual's daughter who was also affected with neurocognitive impairment and epilepsy and carried an additional copy-number variant. The duplicated segment involved all four exons of MECP2, but excluded almost the entire 3' untranslated region (UTR), and the genomic rearrangement resulted in a MECP2-TEX28 fusion gene mRNA transcript. Increased expression of MECP2 and the resulting fusion gene were both confirmed; however, western blot analysis of lysates from lymphoblast cells demonstrated increased MeCP2 protein without evidence of a stable fusion gene protein product. The observations of a mildly affected adult male with a MECP2 duplication and paternal transmission of this

  10. Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

    NASA Technical Reports Server (NTRS)

    Mazumder, B.; Fox, P. L.

    1999-01-01

    Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine

  11. A deletion in the proximal untranslated pX region of human T-cell leukemia virus type II decreases viral replication but not infectivity in vivo.

    PubMed

    Cockerell, G L; Rovnak, J; Green, P L; Chen, I S

    1996-02-01

    The function of untranslated (UT) nucleotide sequences in the proximal portion of the pX region of the human T-cell leukemia virus (HTLV) family of retroviruses remains enigmatic. Previous studies have shown that these sequences are not necessary for the expression of viral proteins or for the induction, transmission, or maintenance of the transformed cell type in vitro. To determine the effect of the UT region in vivo, separate groups of rabbits were inoculated with lethally irradiated, stable clones of the human B-lymphoblastoid cell line, 729, transfected with either a full-length wild-type HTLV-II clone (pH6neo) or a mutant clone containing a 324-bp deletion in the proximal UT portion of pX (pH6neo delta UT[6661-6984]), or nontransfected 729 cells. All rabbits inoculated with either wild-type or pX-deleted HTLV-II developed a similar profile and titer of serum antibodies against HTLV-II antigens, as determined by Western immunoblots, by 4 weeks postinoculation (PI). Antibody titers, as determined by enzyme immunoassay, were similar between the two groups of rabbits and increased over the 18-week period of study. All rabbits were killed at 18 weeks PI, and spleen, peripheral blood lymphocytes (PBMC), bone marrow, and mesenteric lymph node were assayed for HTLV-II tax/rex sequences by quantitative polymerase chain reaction. Virus was detected in all tissues tested from all rabbits inoculated with 729pH6neo cells containing wild-type HTLV-II, which contained between 1.4 and 0.3 mean copies of provirus per cell. In contrast, the distribution and number of provirus copies were more limited in rabbits inoculated with 729pH6neo delta UT(6661-6984) cells containing UT-deleted HTLV-II; in most tissues, there was a fivefold to sevenfold reduction in mean provirus copies per cell as compared with rabbits inoculated with wild-type HTLV-II. All rabbits inoculated with control 729 cells remained negative for HTLV-II infection, as determined by the same techniques. It was

  12. Untranslated region-dependent exclusive expression of high-sensitivity subforms of alpha4beta2 and alpha3beta2 nicotinic acetylcholine receptors.

    PubMed

    Briggs, Clark A; Gubbins, Earl J; Marks, Michael J; Putman, C Brent; Thimmapaya, Rama; Meyer, Michael D; Surowy, Carol S

    2006-07-01

    alpha4beta2 nicotinic acetylcholine receptors (nAChRs) are recognized as the principal nicotine binding site in brain. Recombinant alpha4beta2 nAChR demonstrate biphasic concentration-response relationships with low- and high-EC50 components. This study shows that untranslated regions (UTR) can influence expression of high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChR. Oocytes injected with alpha4 and beta2 RNA lacking UTR expressed biphasic concentration-response relationships for acetylcholine with high-sensitivity EC50 values of 0.5 to 2.5 microM (14-24% of the population) and low-sensitivity EC50 values of 110 to 180 microM (76-86%). In contrast, message with UTR expressed exclusively the high-sensitivity alpha4beta2 nAChR subform with an acetylcholine EC50 value of 2.2 microM. Additional studies revealed pharmacological differences between high- and low-sensitivity alpha4beta2 subforms. Whereas the antagonists dihydro-beta-erythroidine (IC50 of 3-6 nM) and methyllycaconitine (IC50 of 40-135 nM) were not selective between high- and low-sensitivity alpha4beta2, chlorisondamine, mecamylamine, and d-tubocurarine were, respectively, 100-, 8-, and 5-fold selective for the alpha4beta2 subform with low sensitivity to acetylcholine. Conversely, agonists that selectively activated the high-sensitivity alpha4beta2 subform with respect to efficacy as well as potency were identified. Furthermore, two of these agonists were shown to activate mouse brain alpha4beta2 as well as the ferret high-sensitivity alpha4beta2 expressed in Xenopus laevis oocytes. With the use of UTR-containing RNA, exclusive expression of a novel high-sensitivity alpha3beta2 nAChR was also achieved. These studies 1) provide further evidence for the existence of multiple subforms of alpha4beta2 nAChR, 2) extend that to alpha3beta2 nAChR, 3) demonstrate UTR influence on beta2-containing nAChR properties, and 4) reveal compounds that interact with alpha4beta2 in a subform-selective manner.

  13. The 3' untranslated region of the membrane-bound IL-1R accessory protein mRNA confers tissue-specific destabilization.

    PubMed

    Jensen, Liselotte E; Whitehead, Alexander S

    2004-11-15

    IL-1alpha and IL-1beta are proinflammatory cytokines that promote activation of intracellular signaling cascades, leading to stabilization of certain mRNAs and activation of transcription factors. IL-1R type I (IL-1RI) binds IL-1alpha and IL-1beta, and subsequent recruitment of the membrane-bound IL-1R accessory protein (mIL-1RAcP) facilitates signal transduction. Two alternatively spliced isoforms, soluble IL-1RAcP (sIL-1RAcP) and sIL-1RAcP-beta, which lack transmembrane and intracellular domains, have been described. The sIL-1RAcP and possibly sIL-1RAcP-beta can inhibit IL-1 signaling. Proportional expression of the different IL-1RAcP splice variants may be an important determinant of responsiveness to IL-1. We show that although both mIL-1RAcP and sIL-1RAcP mRNAs are widely expressed in human tissue, their relative proportions differ significantly in a tissue-specific manner. Turnover studies revealed that the sIL-1RAcP mRNA has a half-life of approximately 48 h in both the kidney cell line 293 and the hepatoma cell line HepG2. The mIL-1RAcP mRNA has a similar half-life in 293 cells, but a considerably shorter half-life of approximately 5 h in HepG2 cells. Using luciferase reporter constructs, we demonstrated that this specific destabilization of the mIL-1RAcP mRNA in the latter cell type is mediated by its 2.8-kb 3'-untranslated region. Deletion analysis further established that the cell line-specific instability does not involve AU-rich elements, but is mediated by several novel elements that appear to act independently; such elements may be recognized by proteins expressed specifically in some, but not all, tissues. These data demonstrate that the cellular capacity to respond to IL-1 is tightly regulated in a tissue-specific manner.

  14. Novel 5' untranslated region directed blockers of iron-regulatory protein-1 dependent amyloid precursor protein translation: implications for down syndrome and Alzheimer's disease.

    PubMed

    Bandyopadhyay, Sanghamitra; Cahill, Catherine; Balleidier, Amelie; Huang, Conan; Lahiri, Debomoy K; Huang, Xudong; Rogers, Jack T

    2013-01-01

    We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5' untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5'UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the 'ninth' small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5'UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer's disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5'UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5'UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS

  15. A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

    PubMed

    Yoo, Soonmoon; Kim, Hak H; Kim, Paul; Donnelly, Christopher J; Kalinski, Ashley L; Vuppalanchi, Deepika; Park, Michael; Lee, Seung J; Merianda, Tanuja T; Perrone-Bizzozero, Nora I; Twiss, Jeffery L

    2013-09-01

    Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3'UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich regulatory element (ARE) in GAP-43's 3'UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co-immunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3'UTR competing with endogenous GAP-43 mRNA. Conversely, over-expressing GAP-43 coding sequence with its 3'UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43's 3'UTR. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth.

  16. The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

    PubMed Central

    Olivares, Eduardo; Landry, Dori M.; Cáceres, C. Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R.

    2014-01-01

    ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene

  17. Polymorphism in 3' untranslated region of the pig PPARA gene influences its transcript level and is associated with adipose tissue accumulation.

    PubMed

    Stachowiak, M; Szydlowski, M; Flisikowski, K; Flisikowska, T; Bartz, M; Schnieke, A; Switonski, M

    2014-06-01

    The PPARA (peroxisome proliferator-activated receptor-α) gene encodes a nuclear receptor that plays an important role in fatty acid catabolism by transcriptional regulation of genes involved in fatty acid oxidation and can be considered as a candidate gene for fatness traits in the pig. The aim of the study was to search for a functional polymorphism in 3' untranslated region (UTR), their association with production traits, and postnatal PPARA transcript level in 2 skeletal muscles (longissimus and semimembranosus) of 5 commercial pig breeds (Polish Landrace [PL], Polish Large White [PLW], Duroc, Pietrain, and Pulawska). Altogether, 9 novel polymorphisms (8 SNP and 1 indel) were found in the 3' UTR. The in silico analysis revealed 6 putative microRNA target sequences in the analyzed region. The c.*636A>G substitution was widely distributed across breeds and located near the putative target sequence for miR-224. The relative PPARA transcript level was higher (P < 0.05) in LM of AA than in those of GG homozygous animals for SNP c.*636A>G. The luciferase assay revealed that miR-224 probably acts as a negative regulator of the PPARA expression in pig adipocytes (P = 2.9 × 10(-7)), but we did not observe the effect of the A or G alleles on the interaction between miR-224 and its putative target sequence. We hypothesize that the 2 predominant haplotypes, differing at 4 sites (including c.*636A>G), present different architecture of its 3' UTR and it could affect the level of the transcript. The c.*636A>G SNP, analyzed in PL and PLW, was significantly associated with backfat thickness at 3 points (P < 0.05) and intramuscular fat content (P < 0.01) in PL. Suggestive associations were found between 4 SNP (c.*321A>C, c.*324G>C, c.*626T>C, and c.*636A>G) and fatty acid contents in LM and subcutaneous and visceral fat tissue of PL, PLW, Duroc and Pietrain pigs. The PPARA mRNA level was higher in semimembranosus muscle than in LM (P = 8.38 × 10(-12)) in a general comparison

  18. Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5′ Untranslated Region

    PubMed Central

    Thibault, Patricia A.; Huys, Adam; Amador-Cañizares, Yalena; Gailius, Julie E.; Pinel, Dayna E.

    2015-01-01

    ABSTRACT miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5′ untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound full-length RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5′-to-3′ exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122. IMPORTANCE The functions of miR-122 in the promotion of the HCV life cycle are not fully understood. Here, we show that binding of miR-122 to each of the two binding sites in the HCV 5′ UTR contributes equally to HCV replication and that binding to both sites can function cooperatively. This suggests that active Ago2–miR-122 complexes

  19. The 3′ Untranslated Region of Pea Enation Mosaic Virus Contains Two T-Shaped, Ribosome-Binding, Cap-Independent Translation Enhancers

    PubMed Central

    Gao, Feng; Kasprzak, Wojciech K.; Szarko, Christine; Shapiro, Bruce A.

    2014-01-01

    ABSTRACT Many plant viruses without 5′caps or 3′ poly(A) tails contain 3′ proximal, cap-independent translation enhancers (3′CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3′CITEs participate in a long-distance kissing-loop interaction with a 5′ proximal hairpin to deliver ribosomal subunits to the 5′ end for translation initiation. Pea Enation Mosaic Virus (PEMV) contains two adjacent 3′CITEs in the center of its 703-nucleotide 3′ untranslated region (3′UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3′CITE located near the 3′ terminus. This 3′CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3′CITE of the carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3′TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3′TSS contains an apical loop capable of forming a kissing-loop interaction with a 5′ proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3′TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3′UTR in vitro, deleting the normally required kl-TSS and PTE 3′CITEs and placing the kl-H and 3′TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3′CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3′CITE identification. IMPORTANCE The rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3′ cap-independent translation

  20. The proximal region of the 3'-untranslated region of cyclooxygenase-2 is recognized by a multimeric protein complex containing HuR, TIA-1, TIAR, and the heterogeneous nuclear ribonucleoprotein U.

    PubMed

    Cok, Steven J; Acton, Stephen J; Morrison, Aubrey R

    2003-09-19

    Cyclooxygenase-2 (COX-2) is an early response gene induced in renal mesangial cells by interleukin-1beta (IL-1beta). The 3'-untranslated region (3'-UTR) of COX-2 mRNA plays an important role in IL-1beta induction by regulating message stability and translational efficiency. The first 60 nucleotides of the 3'-UTR of COX-2 are highly conserved and contain multiple copies of the regulatory sequence AUUUA. Introduction of the 60-nucleotide sequence into the 3'-UTR of a heterologous reporter gene resulted in a 70% decrease in reporter gene expression. Electrophoretic mobility shift assays (EMSAs) demonstrated that mesangial cell nuclear fractions contain a multimeric protein complex that bound this region of COX-2 mRNA in a sequence-specific manner. We identified four members of the protein-RNA complex as HuR, TIA-1, TIAR, and the heterogeneous nuclear ribonucleoprotein U (hnRNP U). Treatment of mesangial cells with IL-1beta caused an increase in cytosolic HuR, which was accompanied by an increase in COX-2 mRNA that co-immunoprecipitated with cytosolic HuR. Therefore, we propose that HuR binds to the proximal region of the 3'-UTR of COX-2 following stimulation by IL-1beta and increases the expression of COX-2 mRNA by facilitating its transport out of the nucleus.

  1. Comparative analysis of the base compositions of the pre-mRNA 3' cleaved-off region and the mRNA 3' untranslated region relative to the genomic base composition in animals and plants.

    PubMed

    Li, Xiu-Qing

    2014-01-01

    The precursor messenger RNA (pre-mRNA) three-prime cleaved-off region (3'COR) and the mRNA three-prime untranslated region (3'UTR) play critical roles in regulating gene expression. The differences in base composition between these regions and the corresponding genomes are still largely uncharacterized in animals and plants. In this study, the base compositions of non-redundant 3'CORs and 3'UTRs were compared with the corresponding whole genomes of eleven animals, four dicotyledonous plants, and three monocotyledonous (cereal) plants. Among the four bases (A, C, G, and U for adenine, cytosine, guanine, and uracil, respectively), U (which corresponds to T, for thymine, in DNA) was the most frequent, A the second most frequent, G the third most frequent, and C the least frequent in most of the species in both the 3'COR and 3'UTR regions. In comparison with the whole genomes, in both regions the U content was usually the most overrepresented (particularly in the monocotyledonous plants), and the C content was the most underrepresented. The order obtained for the species groups, when ranked from high to low according to the U contents in the 3'COR and 3'UTR was as follows: dicotyledonous plants, monocotyledonous plants, non-mammal animals, and mammals. In contrast, the genomic T content was highest in dicotyledonous plants, lowest in monocotyledonous plants, and intermediate in animals. These results suggest the following: 1) there is a mechanism operating in both animals and plants which is biased toward U and against C in the 3'COR and 3'UTR; 2) the 3'UTR and 3'COR, as functional units, minimized the difference between dicotyledonous and monocotyledonous plants, while the dicotyledonous and monocotyledonous genomes evolved into two extreme groups in terms of base composition.

  2. Comparative Analysis of the Base Compositions of the Pre-mRNA 3′ Cleaved-Off Region and the mRNA 3′ Untranslated Region Relative to the Genomic Base Composition in Animals and Plants

    PubMed Central

    Li, Xiu-Qing

    2014-01-01

    The precursor messenger RNA (pre-mRNA) three-prime cleaved-off region (3′COR) and the mRNA three-prime untranslated region (3′UTR) play critical roles in regulating gene expression. The differences in base composition between these regions and the corresponding genomes are still largely uncharacterized in animals and plants. In this study, the base compositions of non-redundant 3′CORs and 3′UTRs were compared with the corresponding whole genomes of eleven animals, four dicotyledonous plants, and three monocotyledonous (cereal) plants. Among the four bases (A, C, G, and U for adenine, cytosine, guanine, and uracil, respectively), U (which corresponds to T, for thymine, in DNA) was the most frequent, A the second most frequent, G the third most frequent, and C the least frequent in most of the species in both the 3′COR and 3′UTR regions. In comparison with the whole genomes, in both regions the U content was usually the most overrepresented (particularly in the monocotyledonous plants), and the C content was the most underrepresented. The order obtained for the species groups, when ranked from high to low according to the U contents in the 3′COR and 3′UTR was as follows: dicotyledonous plants, monocotyledonous plants, non-mammal animals, and mammals. In contrast, the genomic T content was highest in dicotyledonous plants, lowest in monocotyledonous plants, and intermediate in animals. These results suggest the following: 1) there is a mechanism operating in both animals and plants which is biased toward U and against C in the 3′COR and 3′UTR; 2) the 3′UTR and 3′COR, as functional units, minimized the difference between dicotyledonous and monocotyledonous plants, while the dicotyledonous and monocotyledonous genomes evolved into two extreme groups in terms of base composition. PMID:24941005

  3. Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency.

    PubMed

    Peyvandi, F; Garagiola, I; Palla, R; Marziliano, N; Mannucci, P M

    2005-11-01

    Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.

  4. Binding of DEAD-box helicase Dhh1 to the 5'-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells.

    PubMed

    Zhang, Qianjun; Meng, Xiuhua; Li, Delin; Chen, Shaoyin; Luo, Jianmin; Zhu, Linjie; Singer, Robert H; Gu, Wei

    2017-06-09

    Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The 3' untranslated region of the Andes hantavirus small mRNA functionally replaces the poly(A) tail and stimulates cap-dependent translation initiation from the viral mRNA.

    PubMed

    Vera-Otarola, Jorge; Soto-Rifo, Ricardo; Ricci, Emiliano P; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2010-10-01

    In the process of translation of eukaryotic mRNAs, the 5' cap and the 3' poly(A) tail interact synergistically to stimulate protein synthesis. Unlike its cellular counterparts, the small mRNA (SmRNA) of Andes hantavirus (ANDV), a member of the Bunyaviridae, lacks a 3' poly(A) tail. Here we report that the 3' untranslated region (3'UTR) of the ANDV SmRNA functionally replaces a poly(A) tail and synergistically stimulates cap-dependent translation initiation from the viral mRNA. Stimulation of translation by the 3'UTR of the ANDV SmRNA was found to be independent of viral proteins and of host poly(A)-binding protein.

  6. The 3′ Untranslated Region of the Andes Hantavirus Small mRNA Functionally Replaces the Poly(A) Tail and Stimulates Cap-Dependent Translation Initiation from the Viral mRNA ▿

    PubMed Central

    Vera-Otarola, Jorge; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2010-01-01

    In the process of translation of eukaryotic mRNAs, the 5′ cap and the 3′ poly(A) tail interact synergistically to stimulate protein synthesis. Unlike its cellular counterparts, the small mRNA (SmRNA) of Andes hantavirus (ANDV), a member of the Bunyaviridae, lacks a 3′ poly(A) tail. Here we report that the 3′ untranslated region (3′UTR) of the ANDV SmRNA functionally replaces a poly(A) tail and synergistically stimulates cap-dependent translation initiation from the viral mRNA. Stimulation of translation by the 3′UTR of the ANDV SmRNA was found to be independent of viral proteins and of host poly(A)-binding protein. PMID:20660206

  7. Cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene from the Treacher Collins syndrome candidate region at 5q32-q33.1

    SciTech Connect

    Dixon, J.; Loftus, S.K.; Gladwin, A.J.

    1995-03-20

    Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. Previous studies have shown that the Treacher Collins syndrome locus is flanked by D5S519 and SPARC, and a yeast artificial chromosome contig encompassing this {open_quotes}critical region{close_quotes} has been completed. In the current investigation a cosmid containing D5S519 has been used to screen a human placental cDNA library. This has resulted in the cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene. Two different mRNA species that have identical protein coding sequences but that differ in the size and sequence of the 3{prime} untranslated regions (3{prime}UTR) have been identified. The smaller species has a 3{prime}UTR of 1035 bp, whereas that of the larger is 4878 bp. 24 refs., 3 figs.

  8. cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-Hydroxyacyl-CoA dehydrogenase bifunctional enzyme and localization to chromosome 3q26. 3-3q28: A free left Alu arm is inserted in the 3[prime] noncoding region

    SciTech Connect

    Hoefler, G.; Forstner, M.; Hulla, W.; Hiden, M.; Krisper, P.; Kenner, L.; Zechner, R. ); McGuinness, M.C. ); Ried, T.; Lengauer, C. )

    1994-01-01

    Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme is one of the four enzymes of the peroxisomal, [beta]-oxidation pathway. Here, the authors report the full-length human cDNA sequence and the localization of the corresponding gene on chromosome 3q26.3-3q28. The cDNA sequence spans 3779 nucleotides with an open reading frame of 2169 nucleotides. The tripeptide SKL at the carboxy terminus, known to serve as a peroxisomal targeting signal, is present. DNA sequence comparison of the coding region showed an 80% homology between human and rat bifunctional enzyme cDNA. The 3[prime] noncoding sequence contains 117 nucleotides homologous to an Alu repeat. Based on sequence comparison, they propose that these nucleotides are a free left Alu arm with 86% homology to the Alu-J family. RNA analysis shows one band with highest intensity in liver and kidney. This cDNA will allow in-depth studies of molecular defects in patients with defective peroxisomal bifunctional enzyme. Moreover, it will also provide a means for studying the regulation of peroxisomal [beta]-oxidation in humans. 33 refs., 5 figs.

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

    SciTech Connect

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

    1996-01-01

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

  10. Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

    PubMed Central

    Sztuba-Solinska, Joanna; Teramoto, Tadahisa; Rausch, Jason W.; Shapiro, Bruce A.; Padmanabhan, Radhakrishnan; Le Grice, Stuart F. J.

    2013-01-01

    The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (−) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb2+ ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5′–3′ terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3′ terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5′ DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via ‘flipping’ mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3′ DB and its complementary region PK1 indicated that communication between 5′–3′ terminal regions strongly depends on structure and sequence composition of the 5′ cyclization region. PMID:23531545

  11. Maize Mu transposons are targeted to the 5' untranslated region of the gl8 gene and sequences flanking Mu target-site duplications exhibit nonrandom nucleotide composition throughout the genome.

    PubMed Central

    Dietrich, Charles R; Cui, Feng; Packila, Mark L; Li, Jin; Ashlock, Daniel A; Nikolau, Basil J; Schnable, Patrick S

    2002-01-01

    The widespread use of the maize Mutator (Mu) system to generate mutants exploits the preference of Mu transposons to insert into genic regions. However, little is known about the specificity of Mu insertions within genes. Analysis of 79 independently isolated Mu-induced alleles at the gl8 locus established that at least 75 contain Mu insertions. Analysis of the terminal inverted repeats (TIRs) of the inserted transposons defined three new Mu transposons: Mu10, Mu 11, and Mu12. A large percentage (>80%) of the insertions are located in the 5' untranslated region (UTR) of the gl8 gene. Ten positions within the 5' UTR experienced multiple independent Mu insertions. Analyses of the nucleotide composition of the 9-bp TSD and the sequences directly flanking the TSD reveals that the nucleotide composition of Mu insertion sites differs dramatically from that of random DNA. In particular, the frequencies at which C's and G's are observed at positions -2 and +2 (relative to the TSD) are substantially higher than expected. Insertion sites of 315 RescueMu insertions displayed the same nonrandom nucleotide composition observed for the gl8-Mu alleles. Hence, this study provides strong evidence for the involvement of sequences flanking the TSD in Mu insertion-site selection. PMID:11861572

  12. Specific binding of Fusarium graminearum Hex1 protein to untranslated regions of the genomic RNA of Fusarium graminearum virus 1 correlates with increased accumulation of both strands of viral RNA.

    PubMed

    Son, Moonil; Choi, Hoseong; Kim, Kook-Hyung

    2016-02-01

    The HEX1 gene of Fusarium graminearum was previously reported to be required for the efficient accumulation of Fusarium graminearum virus 1 (FgV1) RNA in its host. To investigate the molecular mechanism underlying the production of FgHEX1 and the replication of FgV1 viral RNA, we conducted electrophoretic mobility shift assays (EMSA) with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that FgHex1 and both the 5'- and 3'-untranslated regions of plus-strand FgV1 RNA formed complexes. To determine whether FgHex1 protein affects FgV1 replication, we quantified accumulation viral RNAs in protoplasts and showed that both (+)- and (-)-strands of FgV1 RNAs were increased in the over-expression mutant and decreased in the deletion mutant. These results indicate that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding to the FgV1 genomic RNA.

  13. Sequence variations of the locus-specific 5' untranslated regions of SLA class I genes and the development of a comprehensive genomic DNA-based high-resolution typing method for SLA-2.

    PubMed

    Choi, H; Le, M T; Lee, H; Choi, M-K; Cho, H-S; Nagasundarapandian, S; Kwon, O-J; Kim, J-H; Seo, K; Park, J-K; Lee, J-H; Ho, C-S; Park, C

    2015-10-01

    The genetic diversity of the major histocompatibility complex (MHC) class I molecules of pigs has not been well characterized. Therefore, the influence of MHC genetic diversity on the immune-related traits of pigs, including disease resistance and other MHC-dependent traits, is not well understood. Here, we attempted to develop an efficient method for systemic analysis of the polymorphisms in the epitope-binding region of swine leukocyte antigens (SLA) class I genes. We performed a comparative analysis of the last 92 bp of the 5' untranslated region (UTR) to the beginning of exon 4 of six SLA classical class I-related genes, SLA-1, -2, -3, -4, -5, and -9, from 36 different sequences. Based on this information, we developed a genomic polymerase chain reaction (PCR) and direct sequencing-based comprehensive typing method for SLA-2. We successfully typed SLA-2 from 400 pigs and 8 cell lines, consisting of 9 different pig breeds, and identified 49 SLA-2 alleles, including 31 previously reported alleles and 18 new alleles. We observed differences in the composition of SLA-2 alleles among different breeds. Our method can be used to study other SLA class I loci and to deepen our knowledge of MHC class I genes in pigs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Functional single nucleotide polymorphism in IL-17A 3' untranslated region is targeted by miR-4480 in vitro and may be associated with age-related macular degeneration.

    PubMed

    Popp, Nicholas A; Yu, Dianke; Green, Bridgett; Chew, Emily Y; Ning, Baitang; Chan, Chi-Chao; Tuo, Jingsheng

    2016-01-01

    Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. Genetic factors contributing to AMD include single nucleotide polymorphisms (SNPs) in immune-related genes including CFH, C2, CFI, C9, and C3, thus implicating these pathways in AMD pathogenesis. MicroRNAs (miRNAs) are powerful regulators of gene expression and execute this function by binding to the 3' untranslated region (3'UTR) of target mRNAs, leading to mRNA degradation. In this study, we searched for the possible association of SNPs in the 3'UTR region of IL-17A, a gene implicated in AMD pathogenesis without any previous SNP association with AMD. Using two independent sample cohorts of Caucasian subjects, six SNPs in the IL-17A 3'-UTR were selected for genotyping based on bioinformatic predictions of the SNP effect on microRNA binding. The SNP rs7747909 was found to be associated with AMD (P < 0.05) in the NEI cohort, using a dominant model logistic regression. Luciferase reporter gene assays and RNA electrophoretic mobility shift assays were performed using ARPE-19 cells to confirm the preferential binding of microRNAs to the major allele of the SNP. Our findings support the hypothesis that microRNA-mediated gene dysregulation may play a role in the pathogenesis of AMD.

  15. p190RhoGEF Binds to a destabilizing element in the 3' untranslated region of light neurofilament subunit mRNA and alters the stability of the transcript.

    PubMed

    Cañete-Soler, R; Wu, J; Zhai, J; Shamim, M; Schlaepfer, W W

    2001-08-24

    Stabilization of neurofilament (NF) mRNAs plays a major role in regulating levels of NF expression and in establishing axonal size and rate of axonal conduction. Previous studies have identified a 68-nucleotide destabilizing element at the junction of the coding region and 3' untranslated region of the light NF subunit (NF-L) mRNA. The present study has used the destabilizing element (probe A) to screen a rat brain cDNA library for interactive proteins. A cDNA clone encoding 1068 nucleotides in the C-terminal domain of p190RhoGEF (clone 39) was found to bind strongly and specifically to the RNA probe. The interaction was confirmed using a glutathione S-transferase/clone 39 fusion protein in Northwestern, gel-shift, and cross-linkage studies. The glutathione S-transferase/clone 39 fusion protein also enhanced the cross-linkage of a major 43-kDa protein in brain extract to the destabilizing element. Functional studies on stably transfected neuronal cells showed that p190RhoGEF expression increased the half-life of a wild-type NF-L mRNA but did not alter the half-life of a mutant NF-L mRNA lacking the destabilizing element. The findings reveal a novel interactive feature of p190RhoGEF that links the exchange factor with NF mRNA stability and regulation of the axonal cytoskeleton.

  16. Sequence and spatial requirements for the tissue- and species-independent 3{prime}-end processing mechanism of plant mRNA

    SciTech Connect

    Wu, L.; Ueda, T.; Messing, J.

    1994-10-01

    Two cis-regulatory regions are required for efficient mRNA 3{prime}-end processing of the maize 27-kDa zein mRNA: a region containing a duplicated AAUGAA poly(A) signal and a region that is present upstream from it. Strict spatial positioning of these two regions is required for efficient mRNA 3{prime}-end processing. Insertions of a stuffer sequence as short as 17 or 18 bp either between the upstream region and the two AAUGAA motifs or between the two AAUGAA motifs drastically reduced the efficiency of 3{prime}-end processing. Mutational analyses of the nucleotide preference at the fourth position of the AAUGAA motif revealed the preference order G > A >> C or U, suggesting that AAUAAA is neither a defective nor an optimal poly(A) signal for the 27-kDa zein mRNA. As for the 3{prime} control region of the cauliflower mosaic virus (CaMV) transcription unit, the mRNA 3{prime}-end processing mechanism mediated by the 27-kDa zein 3{prime} control sequence is neither tissue nor species specific. The 3{prime} upstream sequence of the 27-kDa zein gene can functionally replace that of the CaMV transcription unit. Conversely, the CaMV upstream sequence can mediate mRNA polyadenylation in the presence of a duplicated 27-kDa zein poly(A) signal. However, instead of the proximal poly(A) signal normally used in the 27-kDa zein mRNA, the distal signal is utilized. These results suggest that a general mechanism controls the 3{prime}-end processing of plant mRNAs and that the cis-regulatory functions mediated by their upstream regions are interchangeable. 35 refs., 5 figs.

  17. DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves.

    PubMed

    Safra, Noa; Hayward, Louisa J; Aguilar, Miriam; Sacks, Benjamin N; Westropp, Jodi L; Mohr, F Charles; Mellersh, Cathryn S; Bannasch, Danika L

    2015-01-01

    The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272-422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs.

  18. DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves

    PubMed Central

    Safra, Noa; Hayward, Louisa J.; Aguilar, Miriam; Sacks, Benjamin N.; Westropp, Jodi L.; Mohr, F. Charles; Mellersh, Cathryn S.; Bannasch, Danika L.

    2015-01-01

    The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272–422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs. PMID:26244515

  19. The 3'-untranslated region of alfalfa mosaic virus RNA 3 contains at least two independent binding sites for viral coat protein.

    PubMed Central

    Reusken, C B; Neeleman, L; Bol, J F

    1994-01-01

    The 3'-termini of the three genomic RNAs of alfalfa mosaic virus contain a common sequence of 145 nucleotides (nt) with a specific binding site for coat protein (CP). This sequence consists of several stem/loop structures interspersed with single-stranded AUGC-motifs; in RNA 3 this folding pattern is extended to a region upstream of the homologous sequence. By band-shift assays a minimum of two specific binding sites for CP were identified near the 3'-end of RNA 3. Site 1 consists of the region between nt 11 and 127 from the 3'-end and contains two AUGC-motifs. Site 2 is located between nt 133 and 208 from the 3'-end in a sequence that is largely unique to RNA 3 and contains also two AUGC-motifs. Deletion studies revealed that the two sites could bind CP independently of each other and permitted the identification of sequence elements that are essential for the activity of each site. By site-directed mutagenesis it was shown that the AUGC-motifs are important for binding of CP to both sites. These binding sites may play a role in the phenomenon that each genomic RNA has to be complexed with a few CP molecules to initiate infection. Later in the replication cycle they may act as origins for the assembly of virus particles. Images PMID:8190624

  20. SHAPE Analysis of the RNA Secondary Structure of the Mouse Hepatitis Virus 5′ Untranslated Region and N-Terminal Nsp1 Coding Sequences

    PubMed Central

    Yang, Dong; Liu, Pinghua; Wudeck, Elyse V.; Giedroc, David P.; Leibowitz, Julian L.

    2014-01-01

    SHAPE technology was used to analyze RNA secondary structure of the 5′ most 474 nts of the MHV-A59 genome encompassing the minimal 5′ cis-acting region required for defective interfering RNA replication. The structures generated were in agreement with previous characterizations of SL1 through SL4 and two recently predicted secondary structure elements, S5 and SL5A. SHAPE provided biochemical support for four additional stem-loops not previously functionally investigated in MHV. Secondary structure predictions for 5′ regions of MHV-A59, BCoV and SARS-CoV were similar despite high sequence divergence. The pattern of SHAPE reactivity of in virio genomic RNA, ex virio genomic RNA, and in vitro synthesized RNA were similar, suggesting that binding of N protein or other proteins to virion RNA fails to protect the RNA from reaction with lipid permeable SHAPE reagent. Reverse genetic experiments suggested that SL5C and SL6 within the nsp1 coding sequence are not required for viral replication. PMID:25462342

  1. Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs

    PubMed Central

    Valli, Adrian A.; Santos, Bruno A.C.M.; Hnatova, Silvia; Bassett, Andrew R.; Molnar, Attila; Chung, Betty Y.; Baulcombe, David C.

    2016-01-01

    We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNA-mediated RNA silencing in the model algal species, Chlamydomonas reinhardtii. Among the mutants from this screen, there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon- and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild-type cells were not due to sRNA-targeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing in C. reinhardtii differs from that of higher plants and informs about the evolution and function of this pathway in eukaryotes. PMID:26968199

  2. IL-1beta induces stabilization of IL-8 mRNA in malignant breast cancer cells via the 3' untranslated region: Involvement of divergent RNA-binding factors HuR, KSRP and TIAR.

    PubMed

    Suswam, Esther A; Nabors, L Burt; Huang, Yuanyuan; Yang, Xiuhua; King, Peter H

    2005-03-01

    IL-8 plays an integral role in promoting the malignant phenotype in breast cancer, and its production is directly influenced by inflammatory cytokines in the tumor microenvironment. Here, we show that activation of IL-1beta receptors on malignant HS578t and MDA-MB-231 breast cancer cells strongly induces IL-8 expression and that RNA stabilization is persistently activated at least 12-24 hr after stimulation. SB 203580 and rapamycin reversed the RNA stabilization effect of IL-1beta in a dose-dependent manner, suggesting involvement of the p38/MAP kinase and mTOR pathways. A luciferase reporter assay indicated that the stabilization effect was dependent on cis elements in the 3'-untranslated region (UTR) of the IL-8 transcript. By UV cross-linking, we identified multiple cellular factors that interact with the IL-8 3'UTR, ranging 34-76 kDa. Immunoprecipitation analysis indicated that HuR, KSRP and TIAR bound to one or more loci in the 3'UTR. While the cross-linking patterns were similar, quantitative immunoprecipitation of native IL-8 RNA from IL-1beta-stimulated cytoplasmic extract revealed a 20-fold greater association of transcript with the stabilizing factor HuR vs. the destabilizing factor KSRP. In conclusion, IL-1beta is a potent cytokine stimulus for IL-8 RNA stabilization in breast cancer cells, possibly by enhanced binding of cytoplasmic HuR to the 3'UTR.

  3. microRNA-4717 differentially interacts with its polymorphic target in the PD1 3' untranslated region: A mechanism for regulating PD-1 expression and function in HBV-associated liver diseases.

    PubMed

    Zhang, Guoyu; Li, Na; Li, Zhu; Zhu, Qianqian; Li, Fang; Yang, Cuiling; Han, Qunying; Lv, Yi; Zhou, Zhihua; Liu, Zhengwen

    2015-08-07

    Programmed cell death-1 (PD-1) is involved in hepatitis B virus (HBV) infection, the leading cause of hepatocellular carcinoma (HCC) worldwide. Single-nucleotide polymorphism, rs10204525, located in the PD1 3' untranslated regions (UTR), is associated with chronic HBV infection. MicroRNAs (miRNAs) regulate gene expression via specific binding to the target 3'UTR of mRNA. In this study, three miRNAs were predicted to putatively interact with PD1 rs10204525 polymorphic site of allele G. One of them, miRNA-4717, was demonstrated to allele-specifically affect luciferase activity in a dose-dependent manner in cells transfected with vectors containing different rs10204525 alleles. In lymphocytes from chronic HBV patients withrs10204525 genotype GG, miR-4717 mimics significantly decreased PD-1 expression and increased (TNF)-α and interferon (IFN)-γ production. miR-4717 inhibitor significantly increased PD-1 expression and decreased TNF-α and IFN-γ production although not significantly. In lymphocytes from chronic HBV patients with rs10204525 genotype AA, no similar effects were observed. miR-4717 levels in peripheral lymphocytes from patients with HBV-related chronic hepatitis, cirrhosis and HCC were significantly decreased. In conclusion, miR-4717 may allele-specifically regulate PD-1 expression through interaction with the 3' UTR of PD1 mRNA, leading to the alteration of immune regulation and affecting the susceptibility and disease course of chronic HBV infection.

  4. The mammalian target of rapamycin complex 1 regulates leptin biosynthesis in adipocytes at the level of translation: the role of the 5'-untranslated region in the expression of leptin messenger ribonucleic acid.

    PubMed

    Chakrabarti, Partha; Anno, Takatoshi; Manning, Brendan D; Luo, Zhijun; Kandror, Konstantin V

    2008-10-01

    Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5'-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5'-UTR. We found that the presence of leptin 5'-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5'-terminal oligopyrimidine tract or the 5'-UTR.

  5. Functional comparison of catalase genes in the elimination of photorespiratory H2O2 using promoter- and 3'-untranslated region exchange experiments in the Arabidopsis cat2 photorespiratory mutant.

    PubMed

    Hu, Ye-Qin; Liu, Sheng; Yuan, Hong-Mei; Li, Jing; Yan, Da-Wei; Zhang, Jian-Feng; Lu, Ying-Tang

    2010-10-01

    Photorespiration-associated production of H(2) O(2) accounts for the majority of total H(2) O(2) in leaves of C(3) plants and is mainly eliminated by catalases. In Arabidopsis, lack of CAT2, but not CAT1 or CAT3, results in growth suppression and a marked accumulation of H(2) O(2) in leaves. To evaluate the contribution of individual catalase genes and their promoters to catalase function, we investigated the growth suppression and H(2) O(2) accumulation phenotypes of Arabidopsis derivatives expressing catalase genes from heterologous CAT promoters in a cat2 mutant background. The expression of CAT2 from the CAT2 promoter restored the wild-type phenotype in a cat2-1 mutant, while CAT1 and CAT3 promoter-driven expression of CAT2 did not. Ectopic expression of CAT3 from the CAT2 promoter also restored the normal phenotype, unlike that of CAT1 which required replacement of the CAT1 3'-untranslated region (UTR) with that of CAT2. These results demonstrated that the photorespiratory role of CAT2 is determined mainly by the regulation of its promoter activity. The 3'-UTR of CAT2 was vital for controlling CAT2 protein levels under photorespiratory conditions. Identification of component of heterotetramers catalase isoforms suggested that there is some functional redundancy between CAT2 and CAT1 and CAT3. © 2010 Blackwell Publishing Ltd.

  6. A variant in 3′-untranslated region of KRAS compromises its interaction with hsa-let-7g and contributes to the development of lung cancer in patients with COPD

    PubMed Central

    Hu, Hua; Zhang, Linlin; Teng, Geling; Wu, Yanhua; Chen, Ying

    2015-01-01

    Objective The objective of the present study was to explore the molecular mechanism by which a single nucleotide polymorphism (rs712) interferes with interaction between 3′-untranslated region (3′-UTR) of KRAS and let-7g, and its association with development of lung cancer in the patients with COPD. Materials and methods In this study, we confirmed that KRAS is a target of let-7g in lung cancer cells, and that introduction of rs712 minor allele into 3′-UTR significantly compromised the miRNA/mRNA interaction by using a luciferase reporter system. Additionally, a total of 35 lung tissue samples were obtained (TT:17, TG:12, GG:6), and let-7g and KRAS expression levels were determined. Results We showed that let-7g level was similar between groups, and the concentration of KRAS in GG genotype group was significantly higher than in TT or GT genotype group. Meanwhile, we found COPD patients with GG genotype had significantly higher risk for lung cancer (odds ratio OR =6.83, P=0.0081), compared with TT and GT genotypes. Conclusion Our study demonstrated that KRAS 3′-UTR rs712 polymorphism interfered with miRNA/mRNA interaction, and showed that the minor allele was associated with an elevated risk for development of lung cancer in COPD. PMID:26316738

  7. A variant in 3'-untranslated region of KRAS compromises its interaction with hsa-let-7g and contributes to the development of lung cancer in patients with COPD.

    PubMed

    Hu, Hua; Zhang, Linlin; Teng, Geling; Wu, Yanhua; Chen, Ying

    2015-01-01

    The objective of the present study was to explore the molecular mechanism by which a single nucleotide polymorphism (rs712) interferes with interaction between 3'-untranslated region (3'-UTR) of KRAS and let-7g, and its association with development of lung cancer in the patients with COPD. In this study, we confirmed that KRAS is a target of let-7g in lung cancer cells, and that introduction of rs712 minor allele into 3'-UTR significantly compromised the miRNA/mRNA interaction by using a luciferase reporter system. Additionally, a total of 35 lung tissue samples were obtained (TT:17, TG:12, GG:6), and let-7g and KRAS expression levels were determined. We showed that let-7g level was similar between groups, and the concentration of KRAS in GG genotype group was significantly higher than in TT or GT genotype group. Meanwhile, we found COPD patients with GG genotype had significantly higher risk for lung cancer (odds ratio OR =6.83, P=0.0081), compared with TT and GT genotypes. Our study demonstrated that KRAS 3'-UTR rs712 polymorphism interfered with miRNA/mRNA interaction, and showed that the minor allele was associated with an elevated risk for development of lung cancer in COPD.

  8. AU-rich elements in the mRNA 3'-untranslated region of the rat receptor for advanced glycation end products and their relevance to mRNA stability.

    PubMed

    Caballero, José Juan; Girón, María Dolores; Vargas, Alberto Manuel; Sevillano, Natalia; Suárez, María Dolores; Salto, Rafael

    2004-06-18

    Several putative polyadenylation sequences and an adenylate plus timidylate rich element (ARE) are present at the 3' end of the rat advanced glycation end products receptor (RAGE) gene. Two transcripts are generated by the use of alternative polyadenylation sequences, one containing the ARE sequence in its 3'-untranslated region (3'-UTR). Transfections of CHO-k1 or NRK cells with constructs expressing the 3'-UTRs of the transcripts fused to a green fluorescence protein mRNA show that the ARE sequence has a negative effect on protein expression correlating with a decrease in the amount of mRNA, as shown in CHO-k1 transfected cells. When transfected cells were incubated in the presence of Actinomycin D the amount of fluorescence decreased in cells transfected with the ARE sequence, indicating that this sequence induces lower mRNA stability. Thus, alternative polyadenylation signals and an ARE sequence provide a novel mechanism for the regulation of the rat RAGE gene expression.

  9. Expression of the Helicobacter pylori virulence factor vacuolating cytotoxin A (vac A) is influenced by a potential stem‐loop structure in the 5′ untranslated region of the transcript

    PubMed Central

    Amilon, Karin R.; Letley, Darren P.; Winter, Jody A.; Robinson, Karen

    2015-01-01

    Summary The vacuolating cytotoxin, VacA, is an important virulence factor secreted by the gastric pathogen H elicobacter pylori. Certain vac A genotypes are strongly associated with disease risk, but the association is not absolute. The factors determining vac A gene expression are not fully understood, and the mechanisms of its regulation are elusive. We have identified a potential mRNA stem‐loop forming structure in the 5′ untranslated region (UTR) of the vac A transcript. Using site‐directed mutagenesis, we found that disruption of the stem‐loop structure reduced steady‐state mRNA levels between two‐ and sixfold (P = 0.0005) and decreased mRNA half‐life compared with wild type (P = 0.03). This led to a marked reduction in VacA protein levels and overall toxin activity. Additionally, during stressful environmental conditions of acid pH or high environmental salt concentrations, when general transcription of vac A was decreased or increased respectively, the stabilising effects of the stem‐loop were even more pronounced. Our results suggest that the stem‐loop structure in the vac A 5′ UTR is an important determinant of vac A expression through stabilisation of the vac A mRNA transcript and that the stabilising effect is of particular importance during conditions of environmental stress. PMID:26259667

  10. Metal specificity of an iron-responsive element in Alzheimer's APP mRNA 5'untranslated region, tolerance of SH-SY5Y and H4 neural cells to desferrioxamine, clioquinol, VK-28, and a piperazine chelator.

    PubMed

    Bandyopadhyay, S; Huang, X; Cho, H; Greig, N H; Youdim, M B; Rogers, J T

    2006-01-01

    Iron closely regulates the expression of the Alzheimer's Amyloid Precursor Protein (APP) gene at the level of message translation by a pathway similar to iron control of the translation of the ferritin L- and H mRNAs by Iron-responsive Elements in their 5' untranslated regions (5'UTRs). Using transfection based assays in SH-SY5Y neuroblastoma cells we tested the relative efficiency by which iron, copper and zinc up-regulate IRE activity in the APP 5'UTR. Desferrioxamine (high affinity Fe3+ chelator), (ii) clioquinol (low affinity Fe/Cu/Zn chelator), (iii) piperazine-1 (oral Fe chelator), (iv) VK-28 (oral Fe chelator), were tested for their relative modulation of APP 5' UTR directed translation of a luciferase reporter gene. Iron chelation based therapeutic strategies for slowing the progression of Alzheimer's disease (and other neurological disorders that manifest iron imbalance) are discussed with regard to the relative neural toxic action of each chelator in SH-SY5Y cells and in H4 glioblastoma cells.

  11. Human microRNA-155 on Chromosome 21 Differentially Interacts with Its Polymorphic Target in the AGTR1 3′ Untranslated Region: A Mechanism for Functional Single-Nucleotide Polymorphisms Related to Phenotypes

    PubMed Central

    Sethupathy, Praveen ; Borel, Christelle ; Gagnebin, Maryline ; Grant, Gregory R. ; Deutsch, Samuel ; Elton, Terry S. ; Hatzigeorgiou, Artemis G. ; Antonarakis, Stylianos E. 

    2007-01-01

    Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3′ untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3′ UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21. PMID:17668390

  12. Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes.

    PubMed

    Sethupathy, Praveen; Borel, Christelle; Gagnebin, Maryline; Grant, Gregory R; Deutsch, Samuel; Elton, Terry S; Hatzigeorgiou, Artemis G; Antonarakis, Stylianos E

    2007-08-01

    Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3' untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3' UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21.

  13. A 3′ untranslated region variant in FMR1 eliminates neuronal activity-dependent translation of FMRP by disrupting binding of the RNA-binding protein HuR

    PubMed Central

    Suhl, Joshua A.; Muddashetty, Ravi S.; Anderson, Bart R.; Ifrim, Marius F.; Visootsak, Jeannie; Bassell, Gary J.; Warren, Stephen T.

    2015-01-01

    Fragile X syndrome is a common cause of intellectual disability and autism spectrum disorder. The gene underlying the disorder, fragile X mental retardation 1 (FMR1), is silenced in most cases by a CGG-repeat expansion mutation in the 5′ untranslated region (UTR). Recently, we identified a variant located in the 3′UTR of FMR1 enriched among developmentally delayed males with normal repeat lengths. A patient-derived cell line revealed reduced levels of endogenous fragile X mental retardation protein (FMRP), and a reporter containing a patient 3′UTR caused a decrease in expression. A control reporter expressed in cultured mouse cortical neurons showed an expected increase following synaptic stimulation that was absent when expressing the patient reporter, suggesting an impaired response to neuronal activity. Mobility-shift assays using a control RNA detected an RNA–protein interaction that is lost with the patient RNA, and HuR was subsequently identified as an associated protein. Cross-linking immunoprecipitation experiments identified the locus as an in vivo target of HuR, supporting our in vitro findings. These data suggest that the disrupted interaction of HuR impairs activity-dependent translation of FMRP, which may hinder synaptic plasticity in a clinically significant fashion. PMID:26554012

  14. Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

    PubMed Central

    Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

    2002-01-01

    The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

  15. Differential expression in glioblastoma multiforme and cerebral hemangioblastoma of cytoplasmic proteins that bind two different domains within the 3'-untranslated region of the human glucose transporter 1 (GLUT1) messenger RNA.

    PubMed Central

    Tsukamoto, H; Boado, R J; Pardridge, W M

    1996-01-01

    The glucose transporter 1 (GLUT1) protein is underexpressed in human glioblastoma multiforme and is overexpressed in human cerebral hemangioblastoma. To gain in-sight into possible posttranscriptional mechanisms regulating the expression of the GLUT1 protein in human brain tumors, cytosolic proteins were prepared from these two tumors and used in RNase T1 protection assays that employed [32P]human GLUT1 synthetic RNA prepared from transcription plasmids. Gel shift mobility assays and ultra-violet light cross-linking studies demonstrated the formation of specific RNA/protein complexes that migrated with a mol mass of 120, 44, and 41 kD. RNase T1 mapping and oligodeoxynucleotide competition studies showed that the 120 kD complex was comprised of an RNA fragment that localized to nucleotides 2186-2203 of the GLUT1 mRNA. The 44 kD complex contained an adenosine-uridine-rich RNA fragment that localized to nucleotides 1885-1906 of the human GLUT1 mRNA, and the formation of this complex was inhibited by synthetic RNA enriched in adenosine-uridine sequences. The 44 kD complex was selectively downregulated in hemangioblastoma as compared to glioblastoma multiforme. These studies demonstrate that human brain tumors have differential regulation of cytosolic proteins that specifically interact with two different domains in the 3'-untranslated region of the GLUT1 mRNA, which may serve to mediate the posttranscriptional regulation of GLUT1 gene expression in these tumors. PMID:8675694

  16. Molecular cloning of cDNA for the zeta isoform of the 14-3-3 protein: homologous sequences in the 3'-untranslated region of frog and human zeta isoforms.

    PubMed

    Miura, I; Nakajima, T; Ohtani, H; Kashiwagi, A; Nakamura, M

    1997-10-01

    14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that possess diverse biochemical activities such as regulation of gene transcription, cell proliferation and activation of protein kinase C. At least 7 subtypes (alpha to theta) of 14-3-3 protein are known, but the zeta subtype of this protein has been cloned only in mammals. We cloned the zeta subtype of 14-3-3 protein (14-3-3 zeta) from the frog, Rana rugosa. The sequence encoded 245 amino acids that share 92% identity with rat and bovine 14-3-3 zeta s, and 92% with human phospholipase A2 (PLA2; 14-3-3 zeta). Northern blot analysis revealed a single band of about 1.8 kb in tadpoles at stage 25. The 14-3-3 zeta mRNA level was high in the brain, lung, spleen and kidney, and low in the heart and testis, as opposed to the mRNA level, which was only faintly detected in the liver, pancreas, ovary and muscle. Furthermore, high similarity in the 3'-untranslated region (3'-UTR) was observed between frog and human 14-3-3 zeta cDNA. The results suggest that 14-3-3 zeta is highly conserved throughout eukaryotic evolution, and that the homologous sequence in the 3'-UTR of 14-3-3 zeta cDNA may be conserved in frogs and humans.

  17. The phosphorylation of protein S6 modulates the interaction of the 40 S ribosomal subunit with the 5'-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability.

    PubMed

    Chiaberge, S; Cassarino, E; Mangiarotti, G

    1998-10-16

    AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

  18. Identification of new TSGA10 transcript variants in human testis with conserved regulatory RNA elements in 5'untranslated region and distinct expression in breast cancer.

    PubMed

    Salehipour, Pouya; Nematzadeh, Mahsa; Mobasheri, Maryam Beigom; Afsharpad, Mandana; Mansouri, Kamran; Modarressi, Mohammad Hossein

    2017-09-01

    Testis specific gene antigen 10 (TSGA10) is a cancer testis antigen involved in the process of spermatogenesis. TSGA10 could also play an important role in the inhibition of angiogenesis by preventing nuclear localization of HIF-1α. Although it has been shown that TSGA10 messenger RNA (mRNA) is mainly expressed in testis and some tumors, the transcription pattern and regulatory mechanisms of this gene remain largely unknown. Here, we report that human TSGA10 comprises at least 22 exons and generates four different transcript variants. It was identified that using two distinct promoters and splicing of exons 4 and 7 produced these transcript variants, which have the same coding sequence, but the sequence of 5'untanslated region (5'UTR) is different between them. This is significant because conserved regulatory RNA elements like upstream open reading frame (uORF) and putative internal ribosome entry site (IRES) were found in this region which have different combinations in each transcript variant and it may influence translational efficiency of them in normal or unusual environmental conditions like hypoxia. To indicate the transcription pattern of TSGA10 in breast cancer, expression of identified transcript variants was analyzed in 62 breast cancer samples. We found that TSGA10 tends to express variants with shorter 5'UTR and fewer uORF elements in breast cancer tissues. Our study demonstrates for the first time the expression of different TSGA10 transcript variants in testis and breast cancer tissues and provides a first clue to a role of TSGA10 5'UTR in regulation of translation in unusual environmental conditions like hypoxia. Copyright © 2017. Published by Elsevier B.V.

  19. Tandem repeats 3{prime} of the IGHA genes in the human immunoglobulin heavy chain gene cluster

    SciTech Connect

    Kang, H.K.; Cox, D.W. |

    1996-07-01

    The human IGH constant region spans 350 kb and includes nine genes and two pseudogenes. All of the constant region gene cluster has been cloned except for sequences between the IGHD and IGHG3 genes, between the IGHA1 and IGHA2 gene. The regions 3{prime} of the IGHA genes, which are not cloned, are of interest since transcriptional control elements were found downstream of the IGHA genes in the rat and the mouse IGH loci. In addition, by pulsed-field gel electrophoresis mapping, CpG islands were identified approximately 30 kb downstream of each IGHA gene, within the uncloned portion of the human IGH. These findings indicate that the regions 3{prime} of the IGHA genes to be unclonable by standard cloning methods. Therefore, we applied the Inverse-PCR technique to amplify the sequences flanking the IGHA1 gene. The new sequence included tandem repeats of 20 bp, which we propose is the cause of the unclonability of this region. 39 refs., 6 figs.

  20. Genetic variation in the 3’ untranslated region of dengue virus serotype 3 strains isolated from mosquitoes and humans in Brazil

    PubMed Central

    2013-01-01

    Summary Background Dengue, a mosquito-borne viral infection caused by one of the four dengue virus (DENV) serotypes (DENV-1 to 4), replicate alternately on the mosquito vector and human host and are responsible for infections throughout tropical and subtropical regions of the world. In Brazil, the disease has become a major public health problem and the introduction of DENV-3 in 2000 in Rio de Janeiro (RJ) was associated with severe dengue epidemics. The potential emergence of strains associated with severe disease highlights the need for the surveillance of DENV in human host and vectors. Methods Aiming to contribute for DENV phylogenetic and vector-virus-human host studies, we sequenced the entire genome of one DENV-3 isolated from naturally infected Aedes aegypti from RJ in 2001 and characterized the 3’ UTR from strains isolated from mosquitoes and humans. Mosquitoes were pooled and submitted to virus isolation in Ae. albopictus C6/36 cells and the infecting serotype was identified by immunofluorescence using type-specific monoclonal antibody. Sequence analysis was performed using BioEdit software, the multiple alignments were performed using CLUSTAL W and the phylogenetic analysis by MEGA 5, using the Neighbor-joining method. Secondary structure prediction was performed by using the MFOLD program. Results Exclusive substitutions and a substitution leading to a stop codon on the NS5 gene were observed in the DENV-3 isolated from a naturally infected Ae. aegypti and fully sequenced. As an 8- nucleotides deletion was observed within the 11- nucleotides (nts) insertion on the variable region (VR) from the 3′UTR in this isolate, we further sequenced other DENV-3 from both mosquitoes and humans. The majority of DENV-3 from RJ analyzed were characterized by the 11-nts insertion in the VR of the 3′UTR, despite the observation of strains carrying the 8-nts deletion. The latter presented similar secondary structures, however not all strains presenting the 11-nts

  1. Instability of a dinucleotide repeat in the 3'-untranslated region (UTR) of the microsomal prostaglandin E synthase-1 (mPGES-1) gene in microsatellite instability-high (MSI-H) colorectal carcinoma.

    PubMed

    Cherukuri, Durga Prasad; Deignan, Joshua L; Das, Kingshuk; Grody, Wayne W; Herschman, Harvey

    2015-08-01

    DNA mismatch-repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5'-UTRs and 3'-UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI-H (microsatellite instability-high) tumors. The microsomal prostaglandin E synthase-1 (mPGES-1) gene product, mPGES-1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES-1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES-1 gene 3' untranslated region (3'-UTR), and analyzed its mutation frequencies in MSI-H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI-H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES-1 mRNA levels in MSI-H and MSS colorectal cancer cell lines. Three of four previously designated "MSI-H" cell lines showed higher mPGES-1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES-1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES-1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. The 5' untranslated region of the soybean cytosolic glutamine synthetase β(1) gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants.

    PubMed

    Ortega, Jose Luis; Wilson, Olivia L; Sengupta-Gopalan, Champa

    2012-12-01

    Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In plants, it occurs as two major isoforms, a cytosolic form (GS(1)) and a nuclear encoded chloroplastic form. The focus of this paper is to determine the role of the 5'UTR of a GS(1) gene. GS(1) gene constructs with and without its 5' and 3' UTRs, driven by a constitutive promoter, were agroinfiltrated into tobacco leaves and the tissues were analyzed for both transgene transcript and protein accumulation. The constructs were also tested in an in vitro transcription/translation system and in Escherichia coli. Our results showed that while the 3'UTR functioned in the destabilization of the transcript, the 5'UTR acted as a translation enhancer in plant cells but not in the in vitro translation system. The 5'UTR of the GS(1) gene when placed in front of a reporter gene (uidA), showed a 20-fold increase in the level of GUS expression in agroinfiltrated leaves when compared to the same gene construct without the 5'UTR. The 5'UTR-mediated translational enhancement is probably another step in the regulation of GS in plants. The presence of the GS(1) 5'UTR in front of the GS(1) coding region allowed for its translation in E. coli suggesting the commonality of the translation initiation mechanism for this gene between plants and bacteria.

  3. 5'-Untranslated region of the tryptophan hydroxylase-2 gene harbors an asymmetric bidirectional promoter but not internal ribosome entry site in vitro.

    PubMed

    Chen, Guo-Lin; Miller, Gregory M

    2009-04-15

    Tryptophan hydroxylase-2 (TPH2) catalyzes the synthesis of neuronal serotonin, a major neurotransmitter involved in many brain functions and psychiatric disorders. We have previously revealed a critical role of the human TPH2 (hTPH2) 5'-UTR in gene expression regulation. This study aimed to further characterize mechanism(s) by which the hTPH2 5'-UTR regulates gene expression. An internal ribosome entry site (IRES) activity in hTPH2 5'-UTR was suggested by the conventional bicistronic reporter assay; however, further stringent experiments, including in vitro translation, quantitative real-time PCR, Northern blot, ribonuclease protection assay, and monocistronic reporter assay, demonstrated that the hTPH2 5'-UTR harbors a bidirectional promoter, but not IRES, within its downstream segment (61-141). The antisense promoter is much stronger than the sense promoter, but the strength of both promoters are cell-line dependent, with the highest and lowest activities being observed in HEK-293T and SK-N-MC cells, respectively. In accordance with our previous findings, the upstream segment (1-60) of hTPH2 5'-UTR suppresses the neighboring promoter of both direction, independent of the cell line and its location in the 5'- or 3'-flanking regions of the gene. In summary, this study demonstrates that no IRES but an asymmetric bidirectional promoter is present in the downstream segment of hTPH2 5'-UTR, and this promoter is susceptible to a gene silencing effect caused by the upstream segment (1-60) of hTPH2 5'-UTR. Our findings point to the potential involvement of antisense transcription and non-coding RNA in the regulation of TPH2 gene expression.

  4. Identification and characterization of a 44 kDa protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization.

    PubMed Central

    Geneste, O; Raffalli, F; Lang, M A

    1996-01-01

    Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041-8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces the RNA-binding protein nor affects the poly(A) tail size. SDS/PAGE of the UV-cross-linked RNA-protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The protein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N-ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDa RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail. PMID:8611142

  5. PCK1 is negatively regulated by bta-miR-26a, and a single-nucleotide polymorphism in the 3' untranslated region is involved in semen quality and longevity of Holstein bulls.

    PubMed

    Huang, Jinming; Guo, Fang; Zhang, Zebin; Zhang, Yuanpei; Wang, Xiuge; Ju, Zhihua; Yang, Chunhong; Wang, Changfa; Hou, Minghai; Zhong, Jifeng

    2016-03-01

    Phosphoenolpyruvate carboxykinase 1 (PCK1) is a multi-functional enzyme that plays important roles in physiological processes, including reproduction. We previously reported that the PCK1 transcript has five splice variants; PCK1-AS4, which lacks exon 5, is enriched in the testis of Holstein bulls. In the present study, we profiled select PCK1 transcript variants in the testis, epididymus, and semen of high- and low-performance bulls, and examined the possibility that microRNAs may be involved in single nucleotide polymorphism (SNP)-mediated modulation of PCK1 expression. PCK1-AS4 abundance is not significantly different between high- and low-performance bulls. Luciferase reporter assays, however, showed that bovine PCK1 expression is repressed by bta-miR-26a in HepG2 hepatocyte cells. One SNP (c. + 2183 G > T) at the miRNA-binding site of PCK1 does not influence PCK1 expression, but is associated with elevated ejaculation volume, fresh sperm motility, and genomic estimated breeding value of longevity, as well as with reduced values of composite index and calving ease. Collectively, the identified 3'-untranslated-region SNP variant highlights the importance of PCK1 in the fecundity of Holstein bulls, and implicates a role for bta-miR-26a in regulating PCK1 abundance. Further study is needed to assess the effects of other genetic variants in 5'-flanking region and exons of PCK1 on enzyme levels in the testis and sperm. Mol. Reprod. Dev. 83: 217-225, 2016. © 2016 Wiley Periodicals, Inc.

  6. Next-Generation Sequencing of 5' Untranslated Region of Hepatitis C Virus in Search of Minor Viral Variant in a Patient Who Revealed New Genotype While on Antiviral Treatment.

    PubMed

    Caraballo Cortes, Kamila; Bukowska-Ośko, Iwona; Pawełczyk, Agnieszka; Perlejewski, Karol; Płoski, Rafał; Lechowicz, Urszula; Stawiński, Piotr; Demkow, Urszula; Laskus, Tomasz; Radkowski, Marek

    2016-01-01

    The role of mixed infections with different hepatitis C virus (HCV) genotypes in viral persistence, treatment effects, and tissue tropism is unclear. Next-generation sequencing (NGS), which is suitable for analysis of large, genetically diverse populations offers unparalleled advantages for the study of mixed infections. The aim of the study was to determine, using two different deep sequencing strategies (pyrosequencing - 454 Life Sciences/Roche and reversible terminator sequencing-by-synthesis by Illumina), the origin of a novel HCV genotype transiently detectable during antiviral therapy (pre-existing minor population vs. de novo superinfection). Secondly, we compared 5' untranslated region (5'-UTR) variants obtained by the two NGS approaches. 5' UTR amplification products from 9 samples collected from genotype 1b infected patient before, during, and after treatment (4 serum and 5 peripheral blood mononuclear cell - PBMC - samples) were subjected to the next-generation sequencing. The sequencing revealed the presence of two (454/Roche) and one (Illumina) genotype 4 variants in PBMC at Week 16. None of these variants were present either in the preceding or following samples as revealed by both platforms. 454/Roche sequencing detected 24 different 5'-UTR variants: 8 were present in serum and PBMC, 4 only in serum and 12 only in PBMC. Illumina sequencing detected 11 different 5'-UTR variants: 5 in serum and PBMC, 4 only in serum and 2 only in PBMC. Six variants were identical for both sequencing platforms. The difference in variants number was primarily due to variability in two 5'-UTR homopolymeric regions. In conclusion, longitudinal analysis of HCV variants, employing two independent deep sequencing methods, suggests that the transient presence of a different genotype strain in PBMC was a result of superinfection and not a selection of pre-existing minor variant.

  7. An insertion/deletion polymorphism in the 3' untranslated region of type I collagen a2 (COL1A2) is associated with susceptibility for hepatocellular carcinoma in a Chinese population.

    PubMed

    Zhu, Zhansheng; Jiang, Yuting; Chen, Shougong; Jia, Shasha; Gao, Xueren; Dong, Dong; Gao, Yuzhen

    2011-05-01

    Hepatocellular carcinoma (HCC) is one of the most common and severe diseases in the world. Besides the influence of environmental factors, such as viral infection, an increasing number of novel genetic components identified by genome-wide association studies have been associated with predisposition to HCC. Thus, studies focusing on functional variants in these findings are indispensable. In the present study, based on in-silico analysis, we carried out a case-control study in a Chinese population (207 cases and 245 controls) to investigate the association between HCC susceptibility with a 7 base pair (bp) insertion/deletion polymorphism (rs3917) in the 3'UTR of COL1A2. Our results showed that the ins/del + del/del genotype had an odds ratio of 1.76 (95% C.I.=1.03-3.01; P=0.028) for developing HCC compared to the ins/ins genotype. Carriers for the "del" allele of rs3917 were associated with a 1.73-fold increased risk for HCC (95% C.I.=1.06-2.84; P(trend)=0.02). Computational modeling suggests that this polymorphism is located in the hsa-let-7 g potential target sequence in the COL1A2 3' untranslated region. Our data suggest that most likely, common genetic changes in COL1A2 may influence HCC risk, at least in part by let-7 g-mediated regulation, which is possibly involved in the pathogenesis of HCC. The replication of our studies in other populations will further strengthen our understanding of this association.

  8. Identification of Polymorphisms in the 3′-Untranslated Region of the Human Pregnane X Receptor (PXR) Gene Associated with Variability in Cytochrome P450 3A (CYP3A) Metabolism

    PubMed Central

    Oleson, Lauren; von Moltke, Lisa L.; Greenblatt, David J.; Court, Michael H.

    2013-01-01

    Single nucleotide polymorphisms (SNPs) in the 3′untranslated region (3′UTR) of human pregnane X receptor (PXR) gene may contribute to interindividual variability in cytochrome P450 3A (CYP3A) activity. Genotype-phenotype associations involving PXR-3′UTR SNPs were investigated through in vitro (53 human livers from primarily white donors) and in vivo (26 white or African-American volunteers) studies using midazolam 1′-hydroxylation and midazolam apparent oral clearance (CL/F), respectively, as CYP3A-specific probes. PXR-3′UTR resequencing identified 12 SNPs, including 2 that were novel. Although none of the SNPs evaluated were associated with altered midazolam 1′-hydroxylation in the liver bank, both rs3732359 homozygotes and rs3732360 carriers showed 80% higher (P<0.05) CL/F compared with homozygous reference individuals. These differences in CL/F were even larger (100 and 120% higher, respectively; P<0.01) when only African-American subjects (n=14) were considered. Five major haplotypes were identified containing the PXR-3′UTR SNPs and previously identified intron SNPs. Although CL/F differences were not statistically significant within the entire study cohort, African-American carriers of Haplotype-1 (which includes both rs3732359 and rs3732360 variants) exhibited 70% higher median CL/F compared with African-American non-carriers (P=0.036). Our results identify rs3732359 and rs3732360 as PXR-3′UTR SNPs associated with higher CYP3A activity in vivo in African-Americans. PMID:20082578

  9. Functional polymorphism at the miR-502-binding site in the 3' untranslated region of the SETD8 gene increased the risk of prostate cancer in a sample of Iranian population.

    PubMed

    Narouie, Behzad; Ziaee, Seyed Amir Mohsen; Basiri, Abbas; Hashemi, Mohammad

    2017-08-30

    MicroRNAs (miRNAs), a class of non-coding RNAs, bind to the 3' untranslated regions (3'-UTRs) of target mRNAs and regulate gene expression. Genetic variations in miRNA binding domains influence the susceptibility to several diseases such as cancer. Several studies investigated the impact of single-nucleotide polymorphism (SNP) rs16917496 T>C within the 3'-UTR of SETD8 on cancer susceptibility, but the results were controversial. In addition, no study has been conducted to inspect the impact of this SNP in prostate cancer (PCa). Thus, the present study aimed to find out the possible association between rs16917496 polymorphism at the 3'UTR of SETD8 and PCa risk. This case-control study was done on 169 patients with pathologically confirmed PCa and 182 benign prostatic hyperplasia (BPH). Genotyping was done using PCR-RFLP method. The findings revealed that rs16917496 variant significantly increased the risk of PCa in codominant (OR=2.54, 95%CI=1.50-4.30, p<0.001, TC VS TT and OR=3.03, 95%CI=1.63-5.66, p<0.001, CC vs TT), dominant (OR=2.86, 95%CI=1.62-4.43, p<0.001, p<0.001). The C allele significantly increased the risk of PCa (OR=1.72, 95%CI=1.28-2.33, p<0.001) compared to T allele. In conclusion, the findings indicated that rs16917496 polymorphism may be a risk for predisposition to PCa in an Iranian population. Further studies with larger sample sizes and different ethnicities are required to confirm our findings. Copyright © 2017. Published by Elsevier B.V.

  10. Yersinia enterocolitica Type III Secretion: yscM1 and yscM2 Regulate yop Gene Expression by a Posttranscriptional Mechanism That Targets the 5′ Untranslated Region of yop mRNA

    PubMed Central

    Cambronne, Eric D.; Schneewind, Olaf

    2002-01-01

    Pathogenic Yersinia spp. secrete Yops (Yersinia outer proteins) via the type III pathway. The expression of yop genes is regulated in response to environmental cues, which results in a cascade of type III secretion reactions. yscM1 and yscM2 negatively regulate the expression of Yersinia enterocolitica yop genes. It is demonstrated that yopD and lcrH are required for yscM1 and yscM2 function and that all four genes act synergistically at the same regulatory step. Further, SycH binding to the protein products of yscM1 and yscM2 can activate yop gene expression even without promoting type III transport of YscM1 and YscM2. Reverse transcription-PCR analysis of yopQ mRNA as well as yopQ and yopE gene fusion experiments with the npt (neomycin phosphotransferase) reporter suggest that yscM1 and yscM2 regulate expression at a posttranscriptional step. The 178-nucleotide 5′ untranslated region (UTR) of yopQ mRNA was sufficient to confer yscM1 and yscM2-mediated regulation on the fused reporter, as was the 28-nucleotide UTR of yopE. The sequence 5′-AUAAA-3′ is located in the 5′ yop UTRs, and mutations that alter the sequence motif either reduced or abolished yscM1- and yscM2-mediated regulation. A model is proposed whereby YopD, LcrH, YscM1, YscM2, and SycH regulate yop expression in response to specific environmental cues and by a mechanism that may involve binding of some of these factors to a specific target sequence within the UTR of yop mRNAs. PMID:12374821

  11. XIAP 3'-untranslated region as a ceRNA promotes FSCN1 function in inducing the progression of breast cancer by binding endogenous miR-29a-5p.

    PubMed

    Wu, Qiang; Yan, Hong; Tao, Si-Qi; Wang, Xiao-Nan; Mou, Lang; Chen, Ping; Cheng, Xing-Wang; Wu, Wen-Yong; Wu, Zheng-Sheng

    2017-02-07

    The non-coding 3'-untranslated region (UTR) of genes play an important role in the regulation of microRNA (miRNA) functions, since it can bind and inactivate multiple miRNAs. Herein, we report that ectopic expression of XIAP 3'UTR increased human breast cancer cells proliferation, colony formation, migration, invasion and xenograft tumor growth and suppressed tumor cell death. To investigate this process, we further correlated the genome-wide transcriptional profiling with the gene expression alterations after transfecting XIAP 3'UTR in MCF-7 cells. We identified a robust, genome-wide mechanism of cell migration, motility and epithelial to mesenchymal transition by which mediated by a previously described cellular component movement factor FSCN1. Expression of XIAP and FSCN1 were up-regulated synergistically after transfecting XIAP 3'UTR in vitro and in vivo. Interactions between XIAP and FSCN1 appear to be a key determinant of these processes. Co-transfection with Dicer siRNA reversed the XIAP 3'UTR-mediated oncogenicity, suggesting the miRNAs might be involved in that process. Furthermore, we demonstrated that one miRNA, miR-29a-5p, can bind to both the XIAP and FSCN1 3'UTRs and play an important role in that interactions. We showed that the 3'UTR of XIAP was able to antagonize miR-29a-5p, and resulted in the increased translation of XIAP and FSCN1. Thus, our findings reveal important new insights into how XIAP 3'UTR works, suggesting that the non-coding XIAP 3'UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by which XIAP 3'UTR frees the target mRNAs from being repressed.

  12. Mutant copper-zinc superoxide dismutase associated with amyotrophic lateral sclerosis binds to adenine/uridine-rich stability elements in the vascular endothelial growth factor 3′-untranslated region

    PubMed Central

    Li, Xuelin; Lu, Liang; Bush, Donald J.; Zhang, Xiaowen; Zheng, Lei; Suswam, Esther A.; King, Peter H.

    2009-01-01

    Vascular endothelial growth factor (VEGF) is a neurotrophic factor essential for maintenance of motor neurons. Loss of this factor produces a phenotype similar to amyotrophic lateral sclerosis (ALS). We recently showed that ALS-producing mutations of Cu/Zn-superoxide dismutase (SOD1) disrupt post-transcriptional regulation of VEGF mRNA, leading to significant loss of expression. Mutant SOD1 was present in the ribonucleoprotein complex associated with adenine/uridine-rich elements (ARE) of the VEGF 3′-untranslated region (UTR). Here, we show by electrophoretic mobility shift assay that mutant SOD1 bound directly to the VEGF 3′-UTR with a predilection for AREs similar to the RNA stabilizer HuR. SOD1 mutants A4V and G37R showed higher affinity for the ARE than L38V or G93A. Wild-type SOD1 bound very weakly with an apparent Kd 11- to 72-fold higher than mutant forms. Mutant SOD1 showed an additional lower shift with VEGF ARE that was accentuated in the metal-free state. A similar pattern of binding was observed with AREs of tumor necrosis factor-α and interleukin-8, except only a single shift predominated. Using an ELISA-based assay, we demonstrated that mutant SOD1 competes with HuR and neuronal HuC for VEGF 3′-UTR binding. To define potential RNA-binding domains, we truncated G37R, G93A and wild-type SOD1 and found that peptides from the N-terminal portion of the protein that included amino acids 32-49 could recapitulate the binding pattern of full-length protein. Thus, the strong RNA-binding affinity conferred by ALS-associated mutations of SOD1 may contribute to the post-transcriptional dysregulation of VEGF mRNA. PMID:19196430

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

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

  15. Pyrazole-inducible proteins in DBA/2 mouse liver bind with high affinity to the 3'-untranslated regions of the mRNAs of coumarin hydroxylase (CYP2A5) and c-jun.

    PubMed Central

    Thulke-Gross, M; Hergenhahn, M; Tilloy-Ellul, A; Lang, M; Bartsch, H

    1998-01-01

    An important mechanism in the up-regulation of cytochrome P-450 2A5 (CYP2A5, coumarin hydroxylase, Coh) is the stabilization of the corresponding mRNA; some evidence suggests that proteins binding to CYP2A5 mRNA may be involved in this stabilization. Here we report that pyrazole, a well known inducer of CYP2A5 and stabilizer of its message, enhances the binding of a set of proteins to 32P-labelled 3'-untranslated region (3'UTR) of CYP2A5 to give 32P-labelled bands of apparent molecular mass 37/39, 45/48 and 70/72 kDa after UV cross-linking/RNase cleavage; in addition, we found different proteins binding to other parts of CYP2A5 mRNA. The 70/72 kDa bands are also formed with the 3'UTR of c-jun. The inducible proteins are found in different cellular subfractions at different concentrations, with a maximum of five-fold induction of binding activity in microsomes. When a gel-mobility-shift assay was combined with UV cross-linking to resolve different pyrazole-inducible RNA-protein complexes into single RNA-binding protein bands, the smallest complex contained a double band of 37/39 kDa, 45/48 kDa bands, 70/72 kDa bands, and additional weaker bands at higher molecular masses (around 120 kDa). This composition was found also for all other complexes detected by gel-mobility-shift assay; occasionally, bands at higher molecular masses were also observed. The proteins of the smallest complex might therefore represent a core with which other proteins interact to build up larger complexes. Binding of proteins 37/39 kDa and 70/72 kDa was located to a 20-base loop and adjacent sequences in a 70 nt AU-rich region of the 3'UTR of the CYP2A5. Based on our previous evidence, this 70-nt sequence may play an important role in the stabilization and processing of the message. PMID:9531487

  16. Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins.

    PubMed

    Balmer, L A; Beveridge, D J; Jazayeri, J A; Thomson, A M; Walker, C E; Leedman, P J

    2001-03-01

    The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU

  17. Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5[prime] and 3[prime] Sequences.

    PubMed Central

    Marshall, J. S.; Stubbs, J. D.; Chitty, J. A.; Surin, B.; Taylor, W. C.

    1997-01-01

    The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5[prime] and 3[prime] regions of the Flaveria bidentis Me1 gene fused to the [beta]-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5[prime] region of Me1 determines bundle sheath specificity, whereas the 3[prime] region contains an apparent enhancer-like element that confers high-level expression in leaves. The interaction of 5[prime] and 3[prime] sequences was dependent on factors that are present in the C4 plant but not found in tobacco. PMID:12237394

  18. rs78378222 polymorphism in the 3'-untranslated region of TP53 contributes to development of age-associated cataracts by modifying microRNA-125b-induced apoptosis of lens epithelial cells.

    PubMed

    Zhao, Yang; Li, Xiao; Zhu, Siquan

    2016-09-01

    MicroRNAs (miRNAs) negatively regulate the expression of the target genes by binding to 'seed sequences' in the 3'‑untranslated region (3'‑UTR) mRNA transcripts, and the variants within or nearby 'seed sequences' may compromise or enhance miRNA/mRNA interaction leading to either 'loss‑of‑function' or 'gain‑of‑function' effects. Cataracts are the leading cause of blindness worldwide and are characterized by progressive aggregation and precipitation of lens proteins, and the development of age‑related cataracts is associated with dysregulated cellular activities of lens epithelial cells. Luciferase assays and online miRNA databases were used to validate that tumor protein p53 (TP53) is the target gene of miR‑125b. Furthermore, reverse transcription‑quantitative polymerase chain reaction and western blotting were conducted to detect expression levels of miR‑125b and TP53 in different groups of cells transfected with miR‑125b mimics or inhibitors. In addition, flow cytometry analysis and the MTT assay were conducted to detect the effects of miR‑125b on apoptosis and cell viability. The current study demonstrated that the rs78378222 polymorphism minor allele introduces a novel potential miR‑125b binding site in the TP53 3'‑UTR with a consecutive 8‑bp perfect match, creating a 'gain‑of‑function' variant and affecting the regulation of TP53 expression. A luciferase assay demonstrated that transfection of lens epithelial cells with wild type TP53 3'‑UTR significantly reduced the luciferase activity of the miR‑125b overexpressing cells compared with scramble controls. In addition, the luciferase activity of miR‑125b overexpressing cells transfected with the construct containing the rs78378222 polymorphism minor allele was also reduced compared with cells transfected with the wild type 3'‑UTR. Furthermore, it was demonstrated that the expression level of miR‑125 was comparable in epithelial cells from patients with age

  19. Reverse Stroop Effects with Untranslated Responses

    ERIC Educational Resources Information Center

    Blais, Chris; Besner, Derek

    2006-01-01

    Translation accounts have argued that the presence of a Stroop effect in the context of a nonvocal untranslated response is caused by verbal mediation. In its simplest form, color-labeled buttons are translated into a verbal code that interferes with color responses. On this logic, in the reverse Stroop task (identify the word; ignore the color),…

  20. Reverse Stroop Effects with Untranslated Responses

    ERIC Educational Resources Information Center

    Blais, Chris; Besner, Derek

    2006-01-01

    Translation accounts have argued that the presence of a Stroop effect in the context of a nonvocal untranslated response is caused by verbal mediation. In its simplest form, color-labeled buttons are translated into a verbal code that interferes with color responses. On this logic, in the reverse Stroop task (identify the word; ignore the color),…

  1. Comparative performance evaluation of hepatitis C virus genotyping based on the 5' untranslated region versus partial sequencing of the NS5B region of brazilian patients with chronic hepatitis C

    PubMed Central

    2011-01-01

    Background Genotyping of hepatitis C virus (HCV) has become an essential tool for prognosis and prediction of treatment duration. The aim of this study was to compare two HCV genotyping methods: reverse hybridization line probe assay (LiPA v.1) and partial sequencing of the NS5B region. Methods Plasma of 171 patients with chronic hepatitis C were screened using both a commercial method (LiPA HCV Versant, Siemens, Tarrytown, NY, USA) and different primers targeting the NS5B region for PCR amplification and sequencing analysis. Results Comparison of the HCV genotyping methods showed no difference in the classification at the genotype level. However, a total of 82/171 samples (47.9%) including misclassification, non-subtypable, discrepant and inconclusive results were not classified by LiPA at the subtype level but could be discriminated by NS5B sequencing. Of these samples, 34 samples of genotype 1a and 6 samples of genotype 1b were classified at the subtype level using sequencing of NS5B. Conclusions Sequence analysis of NS5B for genotyping HCV provides precise genotype and subtype identification and an accurate epidemiological representation of circulating viral strains. PMID:21967749

  2. Saccharomyces cerevisiae Hrq1 requires a long 3 Prime -tailed DNA substrate for helicase activity

    SciTech Connect

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Hrq1 has intrinsic 3 Prime -5 Prime helicase and DNA strand annealing activities. Black-Right-Pointing-Pointer Hrq1 requires a long 3 Prime -tail for efficient DNA unwinding. Black-Right-Pointing-Pointer Helicase activity of Hrq1 is stimulated by a fork structure. Black-Right-Pointing-Pointer Hrq1 is a moderately processive helicase. -- Abstract: RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3 Prime -5 Prime helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3 Prime -tail ( Greater-Than-Or-Slanted-Equal-To 70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases.

  3. Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2 prime -chloro-2 prime -deoxyuridine 5 prime -triphosphate: A 3 prime -2 prime hydrogen transfer during the formation of 3 prime -keto-2 prime -deoxyuridine 5 prime -triphosphate

    SciTech Connect

    Ashley, G.W.; Harris, G.; Stubbe, J. )

    1988-10-04

    The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate (C1UTP) into a mixture of 2{prime}-deoxyuridine triphosphate (dUTP) and the unstable product 3{prime}-keto-2{prime}-deoxyuridine triphosphate (3{prime}-keto-dUTP). This ketone can be trapped by reduction with NaBH{sub 4}, producing a 4:1 mixture of xylo-dUTP and dUTP. When (3{prime}-{sup 3}H)C1UTP is treated with enzyme in the presence of NaBH{sub 4}, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the {sup 3}H in C1UTP. Degradation of these isomeric nucleosides has established the location of the {sup 3}H in 3{prime}-keto-dUTP as predominantly 2{prime}(S). The xylo-dU had 98.6% of its label at the 2{prime}(S) position and 1.5% at 2{prime}(R). The isolated dU had 89.6% of its label at 2{prime}(S) and 1.4% at 2{prime}(R), with the remaining 9% label inferred to be at the 3{prime}-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1,000 mixture of dUTP and 3{prime}-keto-dUTP, where the 3{prime}-hydrogen of C1UTP is retained at 3{prime} during production of dUTP and is transferred to 2{prime}(S) during production of 3{prime}-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin are discussed in terms of reductase being a model for the B{sub 12}-dependent rearrangement reactions.

  4. Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region.

    PubMed Central

    Tillmar, Linda; Welsh, Nils

    2002-01-01

    BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway. PMID:12359957

  5. An Autosomal Dominant Cerebellar Ataxia Linked to Chromosome 16q22.1 Is Associated with a Single-Nucleotide Substitution in the 5′ Untranslated Region of the Gene Encoding a Protein with Spectrin Repeat and Rho Guanine-Nucleotide Exchange-Factor Domains

    PubMed Central

    Ishikawa, Kinya; Toru, Shuta; Tsunemi, Taiji; Li, Mingshun; Kobayashi, Kazuhiro; Yokota, Takanori; Amino, Takeshi; Owada, Kiyoshi; Fujigasaki, Hiroto; Sakamoto, Masaki; Tomimitsu, Hiroyuki; Takashima, Minoru; Kumagai, Jiro; Noguchi, Yoshihiro; Kawashima, Yoshiyuki; Ohkoshi, Norio; Ishida, Gen; Gomyoda, Manabu; Yoshida, Mari; Hashizume, Yoshio; Saito, Yuko; Murayama, Shigeo; Yamanouchi, Hiroshi; Mizutani, Toshio; Kondo, Ikuko; Toda, Tatsushi; Mizusawa, Hidehiro

    2005-01-01

    Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. By positional cloning, we have identified the gene strongly associated with a form of degenerative ataxia (chromosome 16q22.1–linked ADCA) that clinically shows progressive pure cerebellar ataxia. Detailed examination by use of audiogram suggested that sensorineural hearing impairment may be associated with ataxia in our families. After restricting the candidate region in chromosome 16q22.1 by haplotype analysis, we found that all patients from 52 unrelated Japanese families harbor a heterozygous C→T single-nucleotide substitution, 16 nt upstream of the putative translation initiation site of the gene for a hypothetical protein DKFZP434I216, which we have called “puratrophin-1” (Purkinje cell atrophy associated protein-1). The full-length puratrophin-1 mRNA had an open reading frame of 3,576 nt, predicted to contain important domains, including the spectrin repeat and the guanine-nucleotide exchange factor (GEF) for Rho GTPases, followed by the Dbl-homologous domain, which indicates the role of puratrophin-1 in intracellular signaling and actin dynamics at the Golgi apparatus. Puratrophin-1—normally expressed in a wide range of cells, including epithelial hair cells in the cochlea—was aggregated in Purkinje cells of the chromosome 16q22.1–linked ADCA brains. Consistent with the protein prediction data of puratrophin-1, the Golgi-apparatus membrane protein and spectrin also formed aggregates in Purkinje cells. The present study highlights the importance of the 5′ untranslated region (UTR) in identification of genes of human disease, suggests that a single-nucleotide substitution in the 5′ UTR could be associated with protein aggregation, and indicates that the GEF protein is associated with cerebellar degeneration in humans. PMID:16001362

  6. An AU-Rich Sequence Element (UUUN[A/U]U) Downstream of the Edited C in Apolipoprotein B mRNA Is a High-Affinity Binding Site for Apobec-1: Binding of Apobec-1 to This Motif in the 3′ Untranslated Region of c-myc Increases mRNA Stability

    PubMed Central

    Anant, Shrikant; Davidson, Nicholas O.

    2000-01-01

    Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a Kd of ∼435 nM. A series of AU-rich templates was used to identify a high-affinity (∼50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3′ untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA

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

    SciTech Connect

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

    1989-08-08

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

  8. 3[prime] end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

    SciTech Connect

    Stern, D.B.; Kindle, K.L. )

    1993-04-01

    The research studied the 3[prime] end maturation of green algae chloroplast atpB mRNA. Most data on transcription termination and 3[prime] end maturation in chloroplasts have been based on in vitro experiments. Newly developed chloroplast transformation techniques have allowed the use of a green algae, Chlamydomonas reinhardtii, to examine chloroplast mRNA 3[prime] end stability determinants and mRNA processing both in vitro and in vivo. The results of this research showed that Chlamydomonas chloroplast protein extracts contain an endonuclease activity that cleaves a synthetic precursor of atpB mRNA 10 nucleotides downstream on the mature 3[prime] end in vitro. Rapid cleavage by this endonuclease is followed by exonucleolytic removal of 10 nucleotides to yield the mature 3[prime] end.

  9. An Analysis of the Effects of Untranslated Behavioral Checklists on the Psychometric Properties of Assessment Centers.

    ERIC Educational Resources Information Center

    Donahue, Lisa M.; And Others

    A field study with 178 candidates for a police promotional examination was conducted to investigate the effects of "untranslated" behavioral checklists on certain psychometric properties of an assessment center. The untranslated checklist used all behavioral responses elicited by the assessment center exercises, not just those that met a…

  10. A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation.

    PubMed Central

    Leathers, V; Tanguay, R; Kobayashi, M; Gallie, D R

    1993-01-01

    Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation. Images PMID:8355685

  11. A T-cell specific transcriptional enhancer element 3 prime of C sub. alpha. in the human T-cell receptor. alpha. locus

    SciTech Connect

    Ho, Icheng; Yang, Lihsuan; Morle, G.; Leiden, J.M. )

    1989-09-01

    A transcriptional enhancer element has been identified 4.5 kilobases 3{prime} of C{sub {alpha}} (constant region {alpha} chain) in the human T-cell receptor (TCR) {alpha}-chain locus. This enhancer is active on both a TCR V{sub {alpha}} (variable region {alpha} chain) promoter and the minimal simian virus 40 promoter in TCR {alpha}/{beta} Jurkat and EL4 cells but is inactive on a V{sub {alpha}} promoter TCR {gamma}/{delta} PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and {kappa}B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR {alpha} gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR {alpha}/{beta} lineage. In addition, the TCR {alpha} enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR {alpha} locus.

  12. Effects of 3,3{prime},4,4{prime}-tetrachlorobiphenyl, 2,3,3{prime},4,4{prime}-phntachlorobiphenyl, and 3,3{prime},4,4{prime},5-pentachlorobiphenyl on the developing chicken embryo when injected prior to incubation

    SciTech Connect

    Powell, D.C.; Aulerich, R.J.; Bursian, S.J.; Stromborg, K.L.

    1996-10-25

    Great Lakes waterbird populations have experienced less-than-expected hatchability of eggs and a greater-than-expected incidence of developmental abnormalities. Such deleterious effects have been attributed to polyhalogenated hydrocarbons such as polychlorinated biphenyls (PCBs). PCBs are of primary concern since they are present in significant quantities in the environment. Specific PCB congeners, 3,3,{prime},4,4{prime},5-pentachlorobiphenyl (IUPAC number 126), 3,3,{prime},4,4{prime}-tetrachlorobiphenyl (IUPAC number 77), and 2,3,3{prime},4,4{prime}-chlorobiphenyl (IUPAC number 105), were injected (singly or in combination) into the yolks of White Leghorn chicken (Gallus domesticus) eggs prior to incubation. Teratogenicity was assessed in dead embryos and in hatchlings. Hatchlings were raised for 3 wk to assess body weight gain and mortality. At the end of the 3-wk period, chicks were subjected to necropsy and the brain, bursa, heart, liver, spleen, and testes were removed and weighed. All 3 congeners caused increased embryo mortality, with approximately 50% mortality occurring at 0.6, 8.8, and 5592 {mu}g/kg egg for congeners 126, 77, and 105, respectively. All three congeners also produced significantly more abnormalities than the vehicle. Chicks from PCB-injected eggs had lower body weights at wk 2 and 3 of age. Congener 126 caused lower relative bursa weights, congener 77 caused greater relative spleen weights and lower relative liver weights, and all three congeners caused relative hear weights top be greater when compared to control. 42 refs., 1 fig., 12 tabs.

  13. Template length, sequence context, and 3 prime -5 prime exonuclease activity modulate replicative bypass of thymine glycol lesions in vitro

    SciTech Connect

    Clark, J.M.; Beardsley G.P. )

    1989-01-24

    cis-Thymine glycol, a product of ionizing radiation damage to DNA, has been introduced quantitatively at a single site into oligonucleotide templates. The ability of DNA polymerases to replicate templates containing thymine glycol was studied by a primer extension assay, and three factors that influence replicative bypass of this lesion in vitro have been identified. These factors include template length, sequence context, and 3{prime}-5{prime} exonuclease activity. Synthesis by the large fragment of DNA polymerase I (Klenow fragment) terminates quantitatively opposite thymine glycol when the template strand extends only two nucleotides beyond the lesion. Significant bypass is observed when the length of the template beyond the lesion is increased to six nucleotides. On the longer templates, the frequency of bypass of the Klenow fragment depends upon the identity of the base immediately 5{prime} to thymine glycol. The extent of bypass is greatest with cytosine and least with adenine at this position. Bypass of thymine glycol lesions by DNA polymerase {alpha}{sub 2} from HeLa cells shows a qualitatively similar dependence upon local sequence context. In contrast, synthesis by T4 DNA polymerase is quantitatively blocked opposite the lesion regardless of template length or DNA sequence context. Synthesis by a mutant Klenow fragment that is deficient in 3{prime}-5{prime} exonuclease activity, or by AMV reverse transcriptase, results in a significant increase in the frequency of bypass. Thus, increased nucleotide turnover at, or beyond, the site of the lesion is likely to contribute significantly to the arrest of synthesis provoked by cis-thymine glycol in vitro.

  14. Trans-activation function of a 3 prime truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues

    SciTech Connect

    Takada, Shinako; Koike, Katsuro )

    1990-08-01

    To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3{prime} end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.

  15. Characterization of the Small Untranslated RNA RyhB and Its Regulon in Vibrio cholerae†

    PubMed Central

    Davis, Brigid M.; Quinones, Mariam; Pratt, Jason; Ding, Yanpeng; Waldor, Matthew K.

    2005-01-01

    Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae, and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli. As in E. coli, V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae. PMID:15937163

  16. Genetic variation at the ApoB 3[prime] HVR, D2S44, and D7S21 loci in the Ewondo ethnic group of Cameroon

    SciTech Connect

    Destro-Bisol, G.; d'Aloja, E.; Dobosz, M.; Pascali, V.L. ); Spedini, G. ); Presciuttini, S. )

    1994-07-01

    A sample of the Ewondo population (a Bantu-speaking group of Southern Cameroon) was analyzed for the polymorphism at three tandem repeated DNA loci (ApoB 3[prime] HVR, D2S44, and D7S21). The authors observed a greater number of ApoB 3[prime] HVR alleles (17) and a significantly higher estimated heterozygosity (.879[+-].011) than in previously surveyed populations, with the exception of U.S. Blacks. The higher genetic variability of Ewondo and U.S. Blacks was also shown by the ApoB 3[prime] HVR allele-frequency spectra. A method for measuring population distances, based on cumulative fragment-size distribution, is described. Interpopulation comparisons for ApoB 3[prime] HVR were carried out by this method and were compared with those obtained by a genetic distance measurement. The two sets of results showed a consistent pattern of population differentiation: the Ewondos and the U.S. Blacks clustered together and were apart from both a Caucasian cluster (Swedes, U.S. Whites, Italians, and Germans) and other well-defined populations (Sikhs of India and Pehuence Indians of Chile). Profile distances were then computed from D2S44 and D7S21 binned data. This analysis indicated a genetic affinity between Ewondos, U.S. Blacks, and Afro-Caribbean Blacks and outlined the genetic diversity between Ewondos, Caucasians, and Asian Indians. 56 refs., 5 figs., 1 tab.

  17. Cortisol metabolism in hepatocytes of rainbow trout treated with 3,3{prime},4,4{prime} tetrachlorobiphenyl

    SciTech Connect

    Vijayan, M.M.; Fiest, G.; Otto, D.; Moon, T.W.

    1995-12-31

    The objective of this study was to investigate the potential of hepatocytes for cortisol uptake and metabolism in 3,3{prime},4,4{prime}-tetrachlorobiphenyl (TCBP) treated trout. Two groups of rainbow trout (Oncorhynchus mykiss) were either given an intraperitoneal implant of peanut oil alone or peanut oil containing TCBP (10 mg.kg{sup {minus}1} body weight) and sampled six weeks later. The toxicant exposed fish had significantly lower condition factor and plasma glucose concentration, whereas plasma cortisol, protein and hepatocyte protein concentration and liver ethoxyresorufin-O-deethylase (EROD) activity were significantly higher in the TCBP compared to the sham group. There was no significant difference in plasma lactate and amino acid concentration, hepatocyte glycogen content or liver cytosolic cortisol binding affinity or capacity between the two groups. The uptake of [{sup 3}H] cortisol was significantly higher in the hepatocytes of TCBP treated fish compared to the sham fish. Also, there was enhanced catabolism of [{sup 3}H] cortisol by hepatocytes of TCBP treated fish; the major metabolite appeared to be tetrahydrocortisone. The results indicate that the potential for cortisol clearance is enhanced in hepatocytes of TCBP treated trout. The data also tend to suggest in vivo regulatory mechanisms that might possibly prevent the increased clearance of the hormone from circulation in toxicant exposed fish.

  18. Subchronic toxicity of 2,2{prime},3,3{prime},4,4{prime}-hexachlorobiphenyl in rats

    SciTech Connect

    Lecavalier, P.; Chu, I.; Feeley, M.

    1997-06-27

    The subchronic toxicity of 2,2{prime},3,3{prime},4,4{prime}-hexachlorobiphenyl (PCB 128) was investigated in rats following dietary exposure at 0, 0.05, 0.5, 5, or 50 ppm for 13 wk. The growth rate was not affected by treatment and no apparent clinical signs of toxicity were observed. There was a significant increase in liver weight in the 50 ppm females. The liver ethoxy-resorufin deethylase (EROD) activity was increased by five- and fourfold in the highest dose males and females, respectively, while aminopyrine demethylase (ADPM) activity was significantly increased only in the highest dose females. Liver vitamin A was significantly reduced in the highest dose females. No other biochemical or hematological effects were observed. Treatment-related histopathological changes were seen in the thyroid and liver, and to a lesser extent in the bone marrow and thymus. Residue data showed a dose-dependent accumulation of PCB 128 in the following tissues: fat, liver, kidney, brain, spleen, and serum, with the highest concentration being found in fat followed by liver and kidney. Based on these data, the no-observable-adverse-effect level of PCB 128 was judged to be 0.5 ppm in diet or 42 {mu}g/kg body weight. 29 refs., 1 fig., 5 tabs.

  19. Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21

    SciTech Connect

    Olopade, O.I.; Pomykala, H.M.; Hagos, F.

    1995-07-03

    Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type I IFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16{sup INK4A}) and CDKN2B (p15{sup INK4B}), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3{prime} untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc{sub 1} complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequences of this gene suggest that it codes for the MTAP enzyme. 35 refs., 4 figs., 1 tab.

  20. Use of 3' untranslated sequences of human cDNAs for rapid chromosome assignment and conversion to STSs: implications for an expression map of the genome.

    PubMed Central

    Wilcox, A S; Khan, A S; Hopkins, J A; Sikela, J M

    1991-01-01

    A general mapping strategy is described in which the 3'untranslated regions of human cDNAs are used to design PCR primers which will selectively amplify human genomic sequences in a rodent background. When applied to panels of human x hamster somatic cell hybrid DNAs, this approach provides a PCR-based method for rapidly assigning genes to specific chromosomes and chromosomal regions. In addition, it follows from the virtual absence of introns in the 3'untranslated region of vertebrate genes that within this region the cDNA sequences almost always will be identical to those of the genomic DNA and can therefore be used to automatically generate gene-specific sequence-tagged sites (STSs). We have applied this strategy to six human cDNAs and demonstrate that 1) the primers selectively amplify human genomic DNA and 2) the PCR product is of the size predicted from the cDNA. To test this approach further we have utilized it to confirm the known chromosomal location of the retinoblastoma gene. Lastly, we describe how this strategy can readily be applied to unknown human cDNAs, and thereby be integrated into efforts to generate a human STS expression map of the genome. A strategy for production of such a map, using human brain cDNAs as a model, is described. Images PMID:2030965

  1. 3' Untranslated regions mediate transcriptional interference between convergent genes both locally and ectopically in Saccharomyces cerevisiae.

    PubMed

    Wang, Luwen; Jiang, Ning; Wang, Lin; Fang, Ou; Leach, Lindsey J; Hu, Xiaohua; Luo, Zewei

    2014-01-01

    Paired sense and antisense (S/AS) genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs) in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3'-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3'-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans) and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the transcriptional collision model, which postulates a clash between simultaneous transcriptional processes occurring on opposite DNA strands.

  2. The role of the 5' untranslated regions of Potyviridae in translation.

    PubMed

    Zhang, Jincan; Roberts, Robyn; Rakotondrafara, Aurélie M

    2015-08-03

    The Potyviridae family relies on a cap-independent translation mechanism to facilitate protein expression. The genomic architecture of the viral RNAs of the Potyviridae family resembles those of the animal picornaviruses. The viral genomes lack a 5' cap structure. Instead, they have the viral protein VPg covalently linked to the 5' end of the RNA. The viral RNAs code for a single large polyprotein, which is then cleaved into several functional subunits. With their common genome organization with the Picornaviridae, it has been largely assumed that the members of the plant Potyviridae family share similar translation mechanism. We will describe the remarkably diverse translational enhancers identified within the family and their unique mechanisms of translation, from internal recruitment of the ribosomes to ribosomal scanning from the 5' end and the recruitment of the VPg in translation. The divergence among the potyviral translation enhancers is heightened with the recent discovery of Triticum mosaic virus, an atypical member of the Potyviridae family, for which its 5' leader by far exceeds the typical length of plant viral leaders and contains features typically found in animal viruses. Much remains to be learned on how these highly divergent elements enable potyviruses, which include some of the most damaging plant viruses, to take over the host translation apparatus. While no clear consensus sequence, structure or mechanism has been reported yet among the potyviral elements, more thorough studies are needed to fill in the gap of knowledge.

  3. Exclusion of candidate genes from the chromosome 1q juvenile glaucoma region and mapping of the peripheral cannabis receptor gene (CNR2) to chromosome 1

    SciTech Connect

    Sunden, S.L.F.; Nichols, B.E.; Alward, W.L.M.

    1994-09-01

    Juvenile onset primary open angle glaucoma has been mapped by linkage to 1q21-q31. Several candidate genes were evaluated in the same family used to identify the primary linkage. Atrionatriuretic peptide receptor A (NPR1) and laminin C1 (LAMC1) have been previously mapped to this region and could putatively play a role in the pathogenesis of glaucoma. A third gene, the peripheral cannabis receptor (CNR2) was not initially mapped in humans but was a candidate because of the relief that cannabis affords some patients with primary open angle glaucoma. Microsatellites associated with NPR1 and LAMC1 revealed multiple recombinations in affected members of this pedigree. CNR2 was shown to be on chromosome 1 by PCR amplification of a 150 bp fragment of the 3{prime} untranslated region in monochromosomal somatic cell hybrids (NIGMS panel No. 2). These primers also revealed a two allele single strand conformation polymorphism which showed multiple recombinants with juvenile onset primary open angle glaucoma in large pedigrees, segregating this disorder. The marker was then mapped to 1p34-p36 by linkage, with the most likely location between liver alkaline phosphatase (ALPL) and alpha-L-1 fucosidase (FUCA1).

  4. An Untranslated cis-Element Regulates the Accumulation of Multiple C4 Enzymes in Gynandropsis gynandra Mesophyll Cells[OPEN

    PubMed Central

    Burgess, Steven J.; Reyna-Llorens, Ivan; Knerova, Jana; Stanley, Susan

    2016-01-01

    C4 photosynthesis is a complex phenotype that allows more efficient carbon capture than the ancestral C3 pathway. In leaves of C4 species, hundreds of transcripts increase in abundance compared with C3 relatives and become restricted to mesophyll (M) or bundle sheath (BS) cells. However, no mechanism has been reported that regulates the compartmentation of multiple enzymes in M or BS cells. We examined mechanisms regulating CARBONIC ANHYDRASE4 (CA4) in C4 Gynandropsis gynandra. Increased abundance is directed by both the promoter region and introns of the G. gynandra gene. A nine-nucleotide motif located in the 5′ untranslated region (UTR) is required for preferential accumulation of GUS in M cells. This element is present and functional in three additional 5′ UTRs and six 3′ UTRs where it determines accumulation of two isoforms of CA and pyruvate,orthophosphate dikinase in M cells. Although the GgCA4 5′ UTR is sufficient to direct GUS accumulation in M cells, transcripts encoding GUS are abundant in both M and BS. Mutating the GgCA4 5′ UTR abolishes enrichment of protein in M cells without affecting transcript abundance. The work identifies a mechanism that directs cell-preferential accumulation of multiple enzymes required for C4 photosynthesis. PMID:26772995

  5. Dynein light chain binding to a 3′-untranslated sequence mediates parathyroid hormone mRNA association with microtubules

    PubMed Central

    Epstein, Eyal; Sela-Brown, Alin; Ringel, Israel; Kilav, Rachel; King, Stephen M.; Benashski, Sharon E.; Yisraeli, Joel K.; Silver, Justin; Naveh-Many, Tally

    2000-01-01

    The 3′-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3′-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3′-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3′-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell. PMID:10683380

  6. 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

  7. Translation of the Saccharomyces cerevisiae tcm1 gene in the absence of a 5'-untranslated leader.

    PubMed Central

    Maicas, E; Shago, M; Friesen, J D

    1990-01-01

    The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other. The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3). Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced. Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet. A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin. This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader. We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency. Images PMID:2216774

  8. VNTR internal structure mapping at the {alpha}-globin 3{prime}HVR locus reveals a hierachy of related lineages in oceania

    SciTech Connect

    Martinson, J.J.; Clegg, J.B.; Boyce, A.J.

    1994-09-01

    Analysis of the {alpha}-globin gene complex in Oceania has revealed many different rearrangements which remove one of the adult globin genes. Frequencies of these deletion chromosomes are elevated by malarial resistance conferred by the resulting {alpha}-thalassaemia. One particular deletion chromosome, designated -{alpha}{sup 3.7}III, is found at high levels in Melanesia and Polynesia: RFLP haplotype analysis shows that this deletion is always found on chromosomes bearing the IIIa haplotype and is likely to be the product of one single rearrangement event. A subset of the -{alpha}{sup 3.7}III chromosomes carries a more recent mutation which generates the haemoglobin variant HbJ{sup Tongariki}. We have characterized the allelic variation at the 3{prime}HVR VNTR locus located 6 kb from the globin genes in each of these groups of chromosomes. We have determined the internal structure of these alleles by RFLP mapping of PCR-amplified DNA: within each group, the allelic diversity results from the insertion and/or deletion of small {open_quotes}motifs{close_quotes} of up to 6 adjacent repeats. Mapping of 3{prime}HVR alleles associated with other haplotypes reveals that these are composed of repeat arrays that are substantially different to those derived from IIIa chromosomes, indicating that interchromosomal recombination between heterologous haplotypes does not account for any of the diversity seen to date. We have recently shown that allelic size variation at the two VNTR loci flanking the {alpha}-globin complex is very closely linked to the haplotypes known to be present at this locus. Here we show that, within a haplotype, VNTR alleles are very closely related to each other on the basis of internal structure and demonstrate that intrachromosomal mutation processes involving small numbers of tandem repeats are the main cause of variation at this locus.

  9. Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression.

    PubMed

    Rosenstiel, Philip; Huse, Klaus; Franke, Andre; Hampe, Jochen; Reichwald, Kathrin; Platzer, Cornelia; Roberts, Roland G; Mathew, Christopher G; Platzer, Matthias; Schreiber, Stefan

    2007-12-20

    NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood. We have identified two novel exons of the NOD2 gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5' untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-alpha or under pro-inflammatory conditions in the inflamed intestinal mucosa in vivo. Using the different 5' UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy. The differential usage of two alternative promoters in the NOD2 gene leads to tissue-specific and context-dependent NOD2 transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling.

  10. Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression

    PubMed Central

    Rosenstiel, Philip; Huse, Klaus; Franke, Andre; Hampe, Jochen; Reichwald, Kathrin; Platzer, Cornelia; Roberts, Roland G; Mathew, Christopher G; Platzer, Matthias; Schreiber, Stefan

    2007-01-01

    Background NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood. Results We have identified two novel exons of the NOD2 gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5' untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under pro-inflammatory conditions in the inflamed intestinal mucosa in vivo. Using the different 5' UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy. Conclusion The differential usage of two alternative promoters in the NOD2 gene leads to tissue-specific and context-dependent NOD2 transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling. PMID:18096043

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

    SciTech Connect

    Williams, C.J.; Ganguly, A.; Considine, E.

    1996-06-14

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

  12. Effects of pre- and postnatal exposure to 3,3[prime],4,4-[prime],5-pentachlorobiphenyl on physical development, neurobehavior and xenobiotic metabolizing enzymes in rats

    SciTech Connect

    Bernhoft, A.; Nafstad, I.; Engen, P. ); Skaare, J.U. )

    1994-10-01

    An experiment was conducted to study the effects of the coplanar non-ortho-chlorinated congener 3,3[prime],4,4[prime],5-pentachlorobiphenyl (PCB-126) in rats exposed during fetal development and postnatal suckling period. Two groups of eight dams were administered by gavage six doses of 10 and 20 [mu]g/kg body weight of PCB-126 dissolved in corn oil every second day from days 9 to 19 of gestation. The corresponding control rats were treated with corn oil only. The physical development of the offspring was observed. The effects of PCB-126 on hepatic xenobiotic metabolizing enzyme activities and the concentrations of PCB in the liver and brain were investigated in samples from pups of different age and from their mothers. The litter size, the body weights, and the survival of the exposed sucklings were reduced, and the onset of spontaneous movement and neuromuscular maturation were delayed, whereas the development of reflexes was not affected. The body weight was still reduced in a dose-related manner up to 18 weeks postpartum. Also, the postpartum body weight of the PCB-exposed mothers was reduced as compared to controls, but the difference disappeared at weaning. The hepatic enzyme activities of cytochrome P450 1A1 examined by ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) toward 1-chloro-2,4-dinitrobenzene (CDNB) were increased in both the exposed pups and their mothers, and the relative liver weight was increased in the exposed pups. Hepatic PCB-126 residues were detected in samples collected throughout the experiment, whereas no detectable concentration was found in the brain. The authors conclude that exposure of this PCB congener in utero and through lactation showed fetotoxic effects, delayed physical maturation, and induced liver xenobiotic metabolizing enzymes without causing neurobehavioral effects.

  13. Mixed-function oxidase enzyme activity and oxidative stress in lake trout (Salvelinus namaycush) exposed to 3,3{prime},4,4{prime}5-pentachlorobiphenyl (PCB-126)

    SciTech Connect

    Palace, V.P.; Klaverkamp, J.F.; Lockhart, W.L. |; Metner, D.A.; Muir, D.C.G.; Brown, S.B.

    1996-06-01

    Juvenile lake trout were intraperitoneally injected with corn oil containing nominal concentrations of 0, 0.6, 6.3, or 25 {micro}g [{sup 14}C]-3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB-126) per gram of body weight. The PCB-126 accumulated in liver in a dose-dependent manner to a sustained concentration by 6 weeks and remained elevated for the 30-week experimental period. Mixed-function oxidase (MFO) enzyme activity was elevated in the two highest dose groups relative to the control group, but not in the low-dose group throughout the 30 weeks. Oxidative stress, measured by the thiobarbituric acid reactive substances test, was correlated with ethoxyresorufin O-deethylase and was elevated in liver of the two highest PCB dose groups but not the low-dose group. The activities of the enzymatic antioxidants superoxide dismutase, catalase, and glutathione peroxidase were unaffected by PCB-126 exposure. The nonenzymatic antioxidants superoxide dismutase, catalase, and glutathione peroxidase were unaffected by PCB-126 exposure. The nonenzymatic antioxidant tocopherol was depleted to approximately 75% of the control concentration in liver of all three PCB-dosed groups. Hepatic ascorbic acid levels were not different in any of the treatment groups. Retinol was depleted by greater than an order of magnitude in liver of the two highest dose groups but not in the los-dose group. This study demonstrates a correlation between hepatic MFO activity and oxidative stress in PCB-exposed lake trout. Tocopherol and retinol may be important mediators of oxidative stress but additional study is required to confirm the antioxidant activity of retinol.

  14. Lack of developmental and reproductive toxicity of 2,3,3{prime},4,4{prime}-pentachlorobiphenyl (PCB 105) in ring-necked pheasants

    SciTech Connect

    Hornung, M.W.; Miller, L.; Peterson, R.E.; Melancon, M.

    1995-12-31

    One of these PCBs, 2,3,3{prime},4,4{prime}-pentachlorobiphenyl (PCB 105) has the potential to produce toxicity by an Ah receptor-mediated mechanism. To determine the potency of PCB 105 for producing reproductive and developmental toxicity, adult ring-necked pheasant hens were orally dosed with 0, 0.06, 0.6 or 6 mg PCB 105/kg hen/week for 10 weeks after which hens were bred with control roosters once per week for 8 weeks. Eggs were collected daily and incubated until hatched, or for 28 days, after which embryo development was evaluated. Fertilized egg production, embryo mortality and chick mortality were not significantly different between treatment groups, nor were total body, liver and heart weights of chicks 1 day post-hatch (dph). To determine whether signs of PCB 105 toxicity were delayed, the first chick to hatch from each hen was evaluated at 21 dph for signs of toxicity. Chick total body, liver and heart weights at 21 dph were not significantly different between treatment groups. Three hepatic microsomal monooxygenase activities were significantly elevated in 1 day old chicks from hens given a cumulative PCB 105 dose of 6 mg/kg and in 21 day old chicks from hens given a cumulative PCB dose of 60 mg/kg as compared to respective control chicks. These results indicate that a cumulative PCB 105 dose up to 60 mg/kg hen does not decrease the production of fertilized eggs or increase embryo or chick mortality in ring-necked pheasants, but does increase chick hepatic monooxygenase activity.

  15. Nuclear Factor E2-Related Factor-2 Negatively Regulates NLRP3 Inflammasome Activity by Inhibiting Reactive Oxygen Species-Induced NLRP3 Priming

    PubMed Central

    Liu, Xiuting; Zhang, Xin; Ding, Yang; Zhou, Wei; Tao, Lei; Lu, Ping; Wang, Yajing

    2017-01-01

    Abstract Aims: The NLRP3 inflammasome is a multiprotein complex that protects hosts against a variety of pathogens. However, the molecular mechanisms of modulating NLRP3 inflammasome activation, especially at the priming step, are still poorly understood. This study was designed to elucidate the negative regulation of nuclear factor E2-related factor-2 (Nrf2) on the activation of NLRP3 inflammasome. Results: We reported that Nrf2 activation inhibited NLRP3 expression, caspase-1 cleavage, and subsequent IL-1β generation. Compared with normal cells, Nrf2-deficient cells showed upregulated cleaved caspase-1, which were attributed to the increased transcription of NLRP3 caused by excess reactive oxygen species (ROS). Furthermore, priming of the NLRP3 inflammasome was sensitive to the exogenous ROS levels induced by H2O2 or rotenone. Combined with adenosine triphosphate, rotenone triggered higher activity of the NLRP3 inflammasome compared with lipopolysaccharide, suggesting that ROS promoted the priming step. In addition, Nrf2-induced NQO1 was involved in the inhibition of the NLRP3 inflammasome. In an in vivo alum-induced peritonitis mouse model, Nrf2 activation suppressed typical IL-1 signaling-dependent inflammation, whereas Nrf2−/− mice exhibited a significant increase in the recruitment of immune cell and the generation of IL-1β compared with wild-type mice. Innovation: We elucidated the effects and possible mechanisms of Nrf2 activation-induced NQO1 expression on NLRP3 inflammasome inactivation and established a novel regulatory role of the Nrf2 pathway in ROS-induced NLRP3 priming. Conclusions: We demonstrated Nrf2 negatively regulating NLRP3 inflammasome activity by inhibiting the priming step and suggested that Nrf2 could be a potential target for some uncontrolled inflammasome activation-associated diseases. Antioxid. Redox Signal. 26, 28–43. PMID:27308893

  16. Tributyltin potentiates 3,3{prime},4,4{prime},5-pentachlorobiphenyl-induced cytochrome P-4501A-related activity

    SciTech Connect

    DeLong, G.T.; Rice, C.D.

    1997-10-01

    Induction of cytochrome P-4501A protein and induction of related enzyme activity are hallmark physiological responses following exposure to planar halogenated aromatic hydrocarbons (HAHs) such as 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126; PeCB). Environments contaminated by HAHs are often contaminated by mixtures of anthropogenic contaminants, including organometallic compounds. Both HAHs and organometallics easily bioconcentrate in aquatic food chains that may be linked to humans through seafood consumption. Tributyltin (TBT), a marine biocide, has been detected in many aquatic environments. Exposure to TBT, as well as several PCBs, has been associated with immunotoxicity, neurotoxicity, and endocrine disruption. Recently TBT has been shown to inhibit cytochrome P-4501A activity in vitro. Female mice were exposed to 0.07, 0.1, and 1.0 mg/kg PeCB, TBT or both. P-4501A levels and BaP-OHase activity were significantly elevated in mice exposed to PeCB alone. This effect was enhanced by coexposure to low levels of TBT; PeCB-induced P-4501A-related activity was potentiated at the low range of each. The highest dose of TBT, however, inhibited these activities when given in combination with PeCB. Thymic atrophy was evident only in mice exposed daily to 0.1 and 1.0 mg/kg PeCB alone, or to a combination of the lowest and highest dose of PeCB and TBT, respectively. Because environmental levels of. TBT are not expected to be as high as the highest level used in our toxicological studies, we conclude that environmental exposure to TBT may potentiate, rather than inhibit, the activity of environmental levels of HAHs that are associated with P-4501A induction. 31 refs., 8 figs.

  17. HPLC determination of tocopherol, retinol, dehydroretinol and retinyl palmitate in tissues of Lake Char (Salvelinus namaycush) exposed to coplanar 3,3[prime],4,4[prime],5-pentachlorobiphenyl

    SciTech Connect

    Palace, V.P. . Dept. of Zoology); Brown, S.B. . Dept. of Fisheries and Oceans)

    1994-03-01

    Tocopherol, retinol, dehydroretinol, and retinyl palmitate were measured by reversed-phase HPLC in liver, kidney, and plasma of lake char exposed to orally administered coplanar 3,3[prime],4,4[prime],5-pentachlorobiphenyl (PCB). Tocopherol concentrations were unaffected after eight weeks. Liver retinol, dehydroretinol, and retinyl palmitate concentrations were lower, whereas kidney retinyl palmitate was elevated in PCB-exposed groups. Tissue retinoid concentrations provide sensitive indicators of coplanar PCB exposure in fish.

  18. Stable, Microfabricated Thin Layer Chromatography Plates without Volume Distortion on Patterned, Carbon and Al2O3-Primed Carbon Nanotube Forests

    SciTech Connect

    Jensen, David S.; Kanyal, Supriya S.; Gupta, Vipul; Vail, Michael A.; Dadson, Andrew; Engelhard, Mark H.; Vanfleet, Richard; Davis, Robert C.; Linford, Matthew R.

    2012-09-28

    In a recent report (Song, J.; et al., Advanced Functional Materials 2011, 21, 1132-1139) some of us described the fabrication of thin layer chromatography (TLC) plates from patterned carbon nanotube (CNT) forests, which were directly infiltrated/coated with silicon by low pressure chemical vapor deposition (LPCVD) of silicon using SiH4. Following infiltration, the nanotubes were removed from the assemblies and the silicon simultaneously converted to SiO2 in a high temperature oxidation step. However, while straightforward, this process had some shortcomings, not the least of which was some distortion of the lithographically patterned features during the volume expansion that accompanied oxidation. Herein we overcome theis issue and also take substantial steps forward in the microfabrication of TLC plates by showing: (i) A new method for creating an adhesion promotion layer on CNT forests by depositing a few nanometers of carbon followed by atomic layer deposition (ALD) of Al2O3. This method for appears to be new, and X-ray photoelectron spectroscopy confirms the expected presence of oxygen after carbon deposition. ALD of Al2O3 alone and in combination with the carbon on patterned CNT forests was also explored as an adhesion promotion layer for CNT forest infiltration. (ii) Rapid, conformal deposition of an inorganic material that does not require subsequent oxidation: fast pseudo-ALD growth of SiO2 via alumina catalyzed deposition of tris(tert-butoxy)silanol onto the carbon/Al2O3-primed CNT forests. (iii) Faithful reproduction of the features in the masks used to microfabricate the TLC plates (M-TLC) this advance springs from the previous two points. (iv) A bonded (amino) phase on a CNT-templated microfabricated TLC plate. (v) Fast, highly efficient (125,000 - 225,000 N/m) separations of fluorescent dyes on M-TLC plates. (vi) Extensive characterization of our new materials by TEM, SEM, EDAX, DRIFT, and XPS. (vii) A substantially lower process temperature for the

  19. 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.

  20. Genetic Variation in the 3'-Untranslated Region of NBN Gene Is Associated with Gastric Cancer Risk in a Chinese Population

    PubMed Central

    Zhu, Xun; Ren, Chuanli; Xie, Lan; Dai, Ningbin; Gu, Yayun; Yan, Caiwang; Dai, Juncheng; Ma, Hongxia; Jiang, Yue; Chen, Jiaping; Hu, Zhibin; Shen, Hongbing; Wu, Haorong; Jin, Guangfu

    2015-01-01

    NBN plays a crucial role in carcinogenesis as a core component for both homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand breaks (DSBs) repair pathways. Genetic variants in the NBN gene have been associated with multiple cancers risk, suggesting pleiotropic effect on cancer. We hypothesized that genetic variants in the NBN gene may modify the risk of gastric cancer. To test this hypothesis, we evaluated the association between four potentially functional single nucleotide polymorphisms in NBN and gastric cancer risk in a case–control study of 1,140 gastric cancer cases and 1,547 controls in a Chinese population. We found that the A allele of rs10464867 (G>A) was significantly associated with a decreased risk of gastric cancer (odds ratio [OR] = 0.81, 95% confidence interval [95% CI] = 0.71–0.94; P = 4.71×10−3). Furthermore, the association between A allele of rs10464867 and decreased risk of gastric cancer was more significantly in elder individuals (per-allele OR = 0.72[0.59–0.88], P = 1.07×10−3), and male individuals (per-allele OR = 0.73[0.62–0.87], P = 3.68×10−4). We further conducted a haplotype analysis and identified that the NBN Ars10464867Grs14448Grs1063053 haplotype conferred stronger protective effect on gastric cancer (OR = 0.76[0.65–0.89], P = 6.39×10−4). In summary, these findings indicate that genetic variants at NBN gene may contribute to gastric cancer susceptibility and may further advance our understanding of NBN gene in cancer development. PMID:26402912

  1. Characterization of different 5'-untranslated exons of the ASIP gene in black-and-tan Doberman Pinscher and brindle Boxer dogs.

    PubMed

    Ciampolini, Roberta; Cecchi, Francesca; Spaterna, Andrea; Bramante, Assunta; Bardet, Sylvia M; Oulmouden, Ahmad

    2013-02-01

    Differential expression of the ASIP gene and its interaction with MC1R have provided basic insight into pigment-type switching in mammals. Here, we report the characterization of a specific red-haired skin transcript and a specific black-haired skin transcript in the ASIP gene in the black-and-tan Doberman Pinscher. It is also shown that the brindle-haired skin of the Boxer exhibits a deregulated expression resulting in various 5'-untranslated exons. Comparative sequence analysis revealed a short interspersed element and a poly(A) stretch inserted within the promoter region of the ASIP in the Boxer. Genotyping studies have shown that both insertions are also present in brindle and fawn animals of the Boxer and Great Dane breeds. Furthermore, we genotyped MC1R and K loci for their known variants that affect coat color in dogs. As expected, all animals were homozygotes (E(M) /E(M) ) for the mask mutation, and fawn animals were k(y) /k(y) . Unexpectedly, we found that all brindle animals were heterozygotes k(B) /k(y) . Our results suggest that differential expression of ASIP determine pigment-type switching in a MC1R and K allele-dependent manner in dogs. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

  2. Effect of 3 prime flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O sup 6 -methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used

    SciTech Connect

    Singer, B.; Chavez, F. ); Goodman, M.F. ); Essigmann, J.M.; Dosanjh, M.K. )

    1989-11-01

    O{sup 6}-Methylguanine (m{sup 6}G) was incorporated site-specifically into two 25-base oligonucleotides differing only in the nucleotide on the 3{prime} side of the modified base. Escherichia coli DNA polymerase I (Klenow fragment) was used to determine the apparent K{sub m} and relative V{sub max} of incorporation of either dCTP or DTTP opposite m{sup 6}G or G. These data were used to calculate the relative frequency of incorporation opposite the m{sup 6}G or the unmodified G. When the sequence was 3{prime}-Cm{sup 6}G-5{prime}, there was a 6- to 7-fold preference for formation of a m{sup 6}G-T pair compared with m{sup 6}G-C. The m{sup 6}G-T frequency, based on V{sub max}/K{sub m}, was at least 50-fold greater than that of a G-T pair at the same site. Changing the sequence to 3{prime}-Tm{sup 6}G-5{prime} had a marked effect on both K{sub m} and V{sub max} of pairs containing m{sup 6}G and on the incorporation frequency of T opposite m{sup 6}G, which was then only slightly favored over m{sup 6}G-C. When replication was started directly opposite m{sup 6}G, the kinetics appeared unaffected. These data indicate that the frequency of incorporation of C or T opposite m{sup 6}G in a DNA template is dependent on the flanking neighbors and that a change of even a single base at the 3{prime} position can have a major effect on mutagenic efficiency. It is concluded that, in the absence of repair, m{sup 6}G can exhibit widely differing mutation frequencies which, in these experiments, can be as high as 85% of the replicated based. This variation in frequency of changed pairing could contribute to the occurrence of mutational hot spots after replication of damage DNA.

  3. Acute toxicity of cadmium, copper, zinc, ammonia, 3,3 prime -dichlorobenzidine, 2,6-dichloro-4-nitroaniline, methylene chloride, and 2,4,6-trichlorophenol to juvenile grass shrimp and killifish

    SciTech Connect

    Burton, D.T.; Fisher, D.J. )

    1990-05-01

    The acute toxicity of several compounds was investigated while performing a toxicity evaluation of a complex chemical effluent. The tests were conducted for one or more of the following reasons: (1) data were not available for the chemical; (2) data were not available for the species; or (3) data were not available for the juvenile life stage of the species. Forty-eight hour acute toxicity tests were run on juvenile grass shrimp (Palaemonetes pugio) and juvenile killifish (Fundulus heteroclitus) exposed to the following compounds: cadmium, copper, zinc, ammonia, 3,3{prime}-dichlorobenzidine, 2,6-dichloro-4-nitroaniline, methylene chloride (dichloromethane) and 2,4,6-trichlorophenol.

  4. Potency of 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126), alone and in combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to produce lake trout early life-stage mortality

    SciTech Connect

    Zabel, E.W.; Peterson, R.E.; Cook, P.M.

    1995-12-01

    Newly fertilized lake trout (Salvelinus namaycush) eggs were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126), or their combination, and sac fry mortality was used to determine toxic potencies. The toxic equivalency factor (TEF) for PCB 126 was 0.0030. The dose-response curve for the PCB 126/TCDD mixture based on TCDD toxic equivalents was not significantly different from that for TCDD alone, suggesting additivity between the two congeners in causing sac fry mortality.

  5. 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.

  6. Sequences of the 5' portion of the human c-sis gene: characterization of the transcriptional promoter and regulation of expression of the protein product by 5' untranslated mRNA sequences.

    PubMed Central

    Ratner, L; Thielan, B; Collins, T

    1987-01-01

    The c-sis gene encodes the B polypeptide chain of platelet-derived growth factor (PDGF), and is expressed in a number of normal and pathological conditions. In order to study the control of synthesis of the human c-sis product, we have initiated a study of two regions of this genetic locus which regulate transcription and translation. A clone of the 5' portion of the gene was obtained which included 1361 nucleotides upstream of the RNA initiation site. Transcriptional promoter activity of this region was demonstrated in normal and transformed cells using a plasmid with the sequences upstream of the c-sis RNA initiation site fused to an indicator gene, chloramphenicol acetyl transferase. Experiments were also performed to identify other possible regulatory regions of the c-sis gene. These data demonstrated that a portion of the c-sis first exon encoding the 5' untranslated region of the c-sis mRNA inhibited synthesis of the PDGF B product in vitro. These results define regions of the c-sis gene whose activity may be important in the regulation of transcription and translation under normal conditions and in the pathogenesis several human diseases. Images PMID:3627977

  7. Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

    PubMed

    Wu, C J; Janssen, G R

    1996-10-01

    The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

  8. Hydrothermal syntheses, crystal structures and luminescence properties of zinc(II) and cadmium(II) coordination polymers based on bifunctional 3,2 Prime :6 Prime ,3 Prime Prime -terpyridine-4 Prime -carboxylic acid

    SciTech Connect

    Li, Na; Guo, Hui-Lin; Hu, Huai-Ming; Song, Juan; Xu, Bing; Yang, Meng-Lin; Dong, Fa-Xin; Xue, Gang-Lin

    2013-02-15

    Five new coordination polymers, [Zn{sub 2}(ctpy){sub 2}Cl{sub 2}]{sub n} (1), [Zn{sub 2}(ctpy){sub 2}(ox)(H{sub 2}O){sub 2}]{sub n} (2), [Zn{sub 2}(ctpy)(3-btc)(H{sub 2}O)]{sub n}{center_dot}0.5nH{sub 2}O (3), [Cd(ctpy){sub 2}(H{sub 2}O)]{sub n} (4), [Cd{sub 4}(ctpy){sub 2}(2-btc){sub 2}(H{sub 2}O){sub 2}]{sub n}{center_dot}2nH{sub 2}O (5), (Hctpy=3,2 Prime :6 Prime ,3 Prime Prime -terpyridine-4 Prime -carboxylic acid, H{sub 2}ox=oxalic acid, H{sub 3}(3-btc)=1,3,5-benzenetricarboxylic acid, H{sub 3}(2-btc)=1,2,4-benzenetricarboxylic acid) have been synthesized under hydrothermal conditions and characterized by elemental analysis, IR spectroscopy, and single-crystal X-ray diffraction. Compounds 1-2 are a one-dimensional chain with weak interactions to form 3D supramolecular structures. Compound 3 is a 4-nodal 3D topology framework comprised of binuclear zinc units and (ctpy){sup -} anions. Compound 4 shows two dimensional net. Compound 5 is a (4,5,6)-connected framework with {l_brace}4{sup 4}{center_dot}6{sup 2}{r_brace}{l_brace}4{sup 6}{center_dot}6{sup 4}{r_brace}{sub 2}{l_brace}4{sup 9}{center_dot}6{sup 6}{r_brace} topology. In addition, the thermal stabilities and photoluminescence properties of 1-5 were also studied in the solid state. - Graphical abstract: Five new Zn/Cd compounds with 3,2 Prime :6 Prime ,3 Prime Prime -terpyridine-4 Prime -carboxylic acid were prepared. The photoluminescence and thermal stabilities properties of 1-5 were investigated in the solid state. Highlights: Black-Right-Pointing-Pointer Five new zinc/cadmium metal-organic frameworks have been hydrothermal synthesized. Black-Right-Pointing-Pointer The structural variation is attributed to the diverse metal ions and auxiliary ligand. Black-Right-Pointing-Pointer Compounds 1-5 exhibit 1D ring chain, 2D layer and 3D open-framework, respectively. Black-Right-Pointing-Pointer These compounds exhibit strong solid state luminescence emission at room temperature.

  9. Pro32Pro33 Mutations in the Integrin β3 PSI Domain Result in αIIbβ3 Priming and Enhanced Adhesion: Reversal of the Hypercoagulability Phenotype by the Src Inhibitor SKI-606

    PubMed Central

    Oliver, Kendra H.; Jessen, Tammy; Crawford, Emily L.; Chung, Chang Y.; Sutcliffe, James S.

    2014-01-01

    The plasma-membrane integrin αIIbβ3 (CD41/CD61, GPIIbIIIa) is a major functional receptor in platelets during clotting. A common isoform of integrin β3, Leu33Pro is associated with enhanced platelet function and increased risk for coronary thrombosis and stroke, although these findings remain controversial. To better understand the molecular mechanisms by which this sequence variation modifies platelet function, we produced transgenic knockin mice expressing a Pro32Pro33 integrin β3. Consistent with reports utilizing human platelets, we found significantly reduced bleeding and clotting times, as well as increased in vivo thrombosis, in Pro32Pro33 homozygous mice. These alterations paralleled increases in platelet attachment and spreading onto fibrinogen resulting from enhanced integrin αIIbβ3 function. Activation with protease-activated receptor 4– activating peptide, the main thrombin signaling receptor in mice, showed no significant difference in activation of Pro32Pro33 mice as compared with controls, suggesting that inside-out signaling remains intact. However, under unstimulated conditions, the Pro32Pro33 mutation led to elevated Src phosphorylation, facilitated by increased talin interactions with the β3 cytoplasmic domain, indicating that the αIIbβ3 intracellular domains are primed for activation while the ligand-binding domain remains unchanged. Acute dosing of animals with a Src inhibitor was sufficient to rescue the clotting phenotype in knockin mice to wild-type levels. Together, our data establish that the Pro32Pro33 structural alteration modifies the function of integrin αIIbβ3, priming the integrin for outside-in signaling, ultimately leading to hypercoagulability. Furthermore, our data may support a novel approach to antiplatelet therapy by Src inhibition where hemostasis is maintained while reducing risk for cardiovascular disease. PMID:24695082

  10. Pro32Pro33 mutations in the integrin β3 PSI domain result in αIIbβ3 priming and enhanced adhesion: reversal of the hypercoagulability phenotype by the Src inhibitor SKI-606.

    PubMed

    Oliver, Kendra H; Jessen, Tammy; Crawford, Emily L; Chung, Chang Y; Sutcliffe, James S; Carneiro, Ana M

    2014-06-01

    The plasma-membrane integrin αIIbβ3 (CD41/CD61, GPIIbIIIa) is a major functional receptor in platelets during clotting. A common isoform of integrin β3, Leu33Pro is associated with enhanced platelet function and increased risk for coronary thrombosis and stroke, although these findings remain controversial. To better understand the molecular mechanisms by which this sequence variation modifies platelet function, we produced transgenic knockin mice expressing a Pro32Pro33 integrin β3. Consistent with reports utilizing human platelets, we found significantly reduced bleeding and clotting times, as well as increased in vivo thrombosis, in Pro32Pro33 homozygous mice. These alterations paralleled increases in platelet attachment and spreading onto fibrinogen resulting from enhanced integrin αIIbβ3 function. Activation with protease-activated receptor 4- activating peptide, the main thrombin signaling receptor in mice, showed no significant difference in activation of Pro32Pro33 mice as compared with controls, suggesting that inside-out signaling remains intact. However, under unstimulated conditions, the Pro32Pro33 mutation led to elevated Src phosphorylation, facilitated by increased talin interactions with the β3 cytoplasmic domain, indicating that the αIIbβ3 intracellular domains are primed for activation while the ligand-binding domain remains unchanged. Acute dosing of animals with a Src inhibitor was sufficient to rescue the clotting phenotype in knockin mice to wild-type levels. Together, our data establish that the Pro32Pro33 structural alteration modifies the function of integrin αIIbβ3, priming the integrin for outside-in signaling, ultimately leading to hypercoagulability. Furthermore, our data may support a novel approach to antiplatelet therapy by Src inhibition where hemostasis is maintained while reducing risk for cardiovascular disease.

  11. Potency of 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126) and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), to produce lake trout early life stage mortality

    SciTech Connect

    Zabel, E.W.; Peterson, R.E.; Cook, P.M.

    1995-12-31

    Toxic equivalency factors (TEFs) relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been determined in rainbow trout for certain polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) using endpoints of early life stage mortality. Rainbow trout are a convenient model fish species. However, one of the major species at risk in the Great Lakes, and the fish species most sensitive to TCDD, is lake trout. The current study sought to (1) determine in lake trout the toxic potency of 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126) in order to test the validity of generalizing TEFs across related species and (2) determine whether PCB 126 and TCDD act in an additive manner at a ratio similar to that found in feral lake trout eggs from the Great Lakes. Newly fertilized lake trout (Salvelinus namaycush) eggs were exposed to TCDD, PCB 126, or their combination at a ratio of 75:1, and sac fry mortality was used to determine toxic potencies. Signs of PCB 126 toxicity in lake trout early life stages were yolk-sac edema, multifocal hemorrhages, craniofacial malformations and mortality, identical to toxicity caused by TCDD. The TEF for PCB 126 was 0.003, which is similar to the TEF of 0.005 determined for rainbow trout early life stage mortality, suggesting that TEFs for the same endpoint are similar across related fish species. The dose-response curve for the congener mixture based on TCDD toxic equivalence was not significantly different from TCDD alone, suggesting that the congener combination acted additively to produce lake trout early life stage mortality.

  12. Chromosomal localization of the human fibromodulin gene

    SciTech Connect

    Roughley, P.J.; Sztrolovics, R.; Grover, J.

    1994-09-01

    The identification and mapping of genes is a fundamental step in understanding inherited diseases. This study reports the chromosomal localization of the human gene encoding fibromodulin, a collagen-binding proteoglycan which exhibits a wide distribution in connective tissue extracellular matrices. Attempts to localize the gene utilizing a probe covering the published coding region of the human fibromodulin cDNA were unsuccessful. Thus, in order to obtain an alternate probe, the 3{prime}-untranslated region of the cDNA was cloned utilizing the 3{prime}-RACE protocol. Southern blot analysis of human genomic DNA with probes covering either the coding sequence or the 3{prime}-untranslated region revealed simple patterns, indicative of a single-copy gene. Fluorescence in situ hybridization analysis with the 3{prime}-untranslated region probe resulted in hybridization at two chromosomal regions. The majority of signals were observed at 1q32, but some signals were also observed at 9q34.1. The localization of the fibromodulin gene to chromosome 1 was confirmed by the polymerase chain reaction analysis of genomic DNA from a panel of somatic cell hybrid lines. In addition to allowing the gene localization, cloning of the 3{prime}-untranslated region demonstrates that the human fibromodulin cDNA possesses an insert of approximately 160 base pairs which is not present in the published bovine sequence. The human sequence also possesses a single polyadenylation signal, yielding a 3 kb mRNA which was observed in Northern blotting experiments. These results now provide the necessary information to evaluate the potential role of fibromodulin in genetic disorders of connective tissues.

  13. TP53 and outcome in DLBCL: not only the coding region.

    PubMed

    Jardin, Fabrice; Coiffier, Bertrand

    2013-05-30

    In this issue of Blood, Li et al report mutations in the 3′ untranslated region (3′UTR) of TP53 that modify the expression of p53 and thus its effect on response to therapy in diffuse large B-cell lymphoma (DLBCL) patients.

  14. Analysis of GNAS1 and overlapping transcripts identifies the parental origin of mutations in patients with sporadic Albright hereditary osteodystrophy and reveals a model system in which to observe the effects of splicing mutations on translated and untranslated messenger RNA.

    PubMed

    Rickard, Sarah J; Wilson, Louise C

    2003-04-01

    Albright hereditary osteodystrophy (AHO) is caused by heterozygous deactivating GNAS1 mutations. There is a parent-of-origin effect. Maternally derived mutations are usually associated with resistance to parathyroid hormone termed "pseudohypoparathyroidism type Ia." Paternally derived mutations are associated with AHO but usually normal hormone responsiveness, known as "pseudo-pseudohypoparathyroidism." These observations can be explained by tissue-specific GNAS1 imprinting. Regulation of the genomic region that encompasses GNAS1 is complex. At least three upstream exons that splice to exon 2 of GNAS1 and that are imprinted have been reported. NESP55 is exclusively maternally expressed, whereas exon 1A and XL alphas are exclusively paternally expressed. We set out to identify the parental origin of GNAS1 mutations in patients with AHO by searching for their mutation in the overlapping transcripts. This information would be of value in patients with sporadic disease, for predicting their endocrine phenotype and planning follow-up. In doing so, we identified mutations that resulted in nonsense-mediated decay of the mutant Gs alpha transcript but that were detectable in NESP55 messenger RNA (mRNA), probably because they lie within its 3' untranslated region. Analysis of the NESP55 transcripts revealed the creation of a novel splice site in one patient and an unusual intronic mutation that caused retention of the intron in a further patient, neither of which could be detected by analysis of the Gs alpha complementary DNA. This cluster of overlapping transcripts represents a useful model system in which to analyze the effects that mutant sequence has on mRNA-in particular, splicing-and the mechanisms of nonsense-mediated mRNA decay.

  15. Analysis of GNAS1 and Overlapping Transcripts Identifies the Parental Origin of Mutations in Patients with Sporadic Albright Hereditary Osteodystrophy and Reveals a Model System in Which to Observe the Effects of Splicing Mutations on Translated and Untranslated Messenger RNA

    PubMed Central

    Rickard, Sarah J.; Wilson, Louise C.

    2003-01-01

    Albright hereditary osteodystrophy (AHO) is caused by heterozygous deactivating GNAS1 mutations. There is a parent-of-origin effect. Maternally derived mutations are usually associated with resistance to parathyroid hormone termed “pseudohypoparathyroidism type Ia.” Paternally derived mutations are associated with AHO but usually normal hormone responsiveness, known as “pseudo-pseudohypoparathyroidism.” These observations can be explained by tissue-specific GNAS1 imprinting. Regulation of the genomic region that encompasses GNAS1 is complex. At least three upstream exons that splice to exon 2 of GNAS1 and that are imprinted have been reported. NESP55 is exclusively maternally expressed, whereas exon 1A and XLαs are exclusively paternally expressed. We set out to identify the parental origin of GNAS1 mutations in patients with AHO by searching for their mutation in the overlapping transcripts. This information would be of value in patients with sporadic disease, for predicting their endocrine phenotype and planning follow-up. In doing so, we identified mutations that resulted in nonsense-mediated decay of the mutant Gsα transcript but that were detectable in NESP55 messenger RNA (mRNA), probably because they lie within its 3′ untranslated region. Analysis of the NESP55 transcripts revealed the creation of a novel splice site in one patient and an unusual intronic mutation that caused retention of the intron in a further patient, neither of which could be detected by analysis of the Gsα complementary DNA. This cluster of overlapping transcripts represents a useful model system in which to analyze the effects that mutant sequence has on mRNA—in particular, splicing—and the mechanisms of nonsense-mediated mRNA decay. PMID:12624854

  16. HLA-G 3'-untranslated region polymorphisms are associated with HTLV-1 infection, proviral load and HTLV-associated myelopathy/tropical spastic paraparesis development.

    PubMed

    Cilião Alves, Daiani Cristina; Haddad, Rodrigo; Rocha-Júnior, Maurício Cristiano; de Deus Wagatsuma, Virgínia Mara; Martelli-Palomino, Gustavo; Marques, Adriana Aparecida; Takayanagui, Osvaldo Massaiti; Covas, Dimas Tadeu; Kashima, Simone; Donadi, Eduardo Antônio

    2016-10-01

    Most human T-lymphotropic virus type 1 (HTLV-1)-infected patients remain asymptomatic throughout life. The factors associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development have not been fully elucidated; immunological and genetic factors may be involved. The association of 14 bp INS/DEL HLA-G polymorphism with HTLV-1 infection susceptibility has been reported previously. Here, other polymorphic sites at the HLA-G 3'-UTR (14-bp D/I, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G and +3196C/G) were evaluated in 37 HTLV-1-infected individuals exhibiting HAM/TSP, 45 HTLV-1 asymptomatic carriers (HAC) and 153 uninfected individuals, followed up at University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. It was observed that: (i) 14bpDI genotype is a risk factor for HTLV-1 infection, while the 14bpDD and +3142CC genotypes were associated with protection against infection; (ii) the +3142C allele and the +3003CT and +3142CC genotypes were associated with susceptibility, while 14bpII and +3003TT genotypes were associated with protection against HAM/TSP development; and (iii) the 14bpII, +3010CC, +3142GG and +3187AA genotypes were associated with lower HTLV-1 proviral load compared to respective counterpart genotypes. Findings that HLA-G has a well-recognized immunomodulatory role and that the genetic variability at HLA-G 3'-UTR may post-transcriptionally modify HLA-G production indicate a differential genetic susceptibility to: (i) the development of HTLV-1 infection, (ii) the magnitude of HTLV-1 proviral load and (iii) HAM/TSP development.

  17. Genome-wide association identifies a deletion in the 3’ untranslated region of Striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy

    USDA-ARS?s Scientific Manuscript database

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by rapid ventricular tachycardia and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. Th...

  18. Identification and characterization of upstream open reading frames (uORF) in the 5' untranslated regions (UTR) of genes in Saccharomyces cerevisiae.

    PubMed

    Zhang, Zhihong; Dietrich, Fred S

    2005-08-01

    We have taken advantage of recently sequenced hemiascomycete fungal genomes to computationally identify additional genes potentially regulated by upstream open reading frames (uORFs). Our approach is based on the observation that the structure, including the uORFs, of the post-transcriptionally uORF regulated Saccharomyces cerevisiae genes GCN4 and CPA1 is conserved in related species. Thirty-eight candidate genes for which uORFs were found in multiple species were identified and tested. We determined by 5' RACE that 15 of these 38 genes are transcribed. Most of these 15 genes have only a single uORF in their 5' UTR, and the length of these uORFs range from 3 to 24 codons. We cloned seven full-length UTR sequences into a luciferase (LUC) reporter system. Luciferase activity and mRNA level were compared between the wild-type UTR construct and a construct where the uORF start codon was mutated. The translational efficiency index (TEI) of each construct was calculated to test the possible regulatory function on translational level. We hypothesize that uORFs in the UTR of RPC11, TPK1, FOL1, WSC3, and MKK1 may have translational regulatory roles while uORFs in the 5' UTR of ECM7 and IMD4 have little effect on translation under the conditions tested.

  19. 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

  20. Variation in the coding and 3’ untranslated regions of the porcine prolactin receptor short form modifies protein expression and function

    USDA-ARS?s Scientific Manuscript database

    The actions of prolactin (PRL) are mediated by both long (LF) and short isoforms (SF) of the PRL receptor (PRLR). Here, we report on a genetic and functional analysis of the porcine PRLR (pPRLR) SF. Three single nucleotide polymorphisms (SNPs) within exon 11 of the pPRLR-SF give rise to four amino a...

  1. A stem–loop structure in the 59 untranslated region of bean pod mottle virus RNA2 is specifically required for RNA2 accumulation

    USDA-ARS?s Scientific Manuscript database

    Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family Secoviridae. Its RNA1 encodes all proteins needed for genome replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought ...

  2. Cooperative enhancement of translation by two adjacent microRNA-122/Argonaute 2 complexes binding to the 5' untranslated region of hepatitis C virus RNA.

    PubMed

    Nieder-Röhrmann, Anika; Dünnes, Nadia; Gerresheim, Gesche K; Shalamova, Lyudmila A; Herchenröther, Andreas; Niepmann, Michael

    2017-02-01

    The liver-specific microRNA-122 (miR-122) binds to two conserved binding sites in the 5' UTR of hepatitis C virus (HCV) RNA. This binding was reported to enhance HCV RNA replication, translation and stability. We have analysed binding of miR-122/Argonaute 2 (Ago2) complexes to these sites using anti-Ago2 co-immunoprecipitation of radioactively labelled HCV RNAs along with ectopic miR-122 in HeLa cells. Our results show that the miR-122 target sites can be addressed separately. When both target sites were addressed simultaneously, we observed a synergistic binding of both miR/Ago2 complexes. Consistently, simultaneous binding of both miR-122/Ago2 complexes results in cooperative translation stimulation. In the binding assays as well as in the translation assays, binding site 1 has a stronger effect than binding site 2. We also analysed the overall RNA stability as well as the 5' end integrity of these HCV RNAs in the presence of miR-122. Surprisingly, using short HCV reporter RNAs, we did not find effects of miR-122 binding on overall RNA stability or 5' end integrity over up to 36 h. In contrast, using full-length HCV genomes that are incapable of replication, we found a positive influence of miR-122 on RNA stability, indicating that features of the full-length HCV genome that do not reside in the 5' and 3' UTRs may render HCV RNA genome stability miR-122 dependent.

  3. Genetic variant in the 3'-untranslated region of VEGFR1 gene influences chronic obstructive pulmonary disease and lung cancer development in Chinese population.

    PubMed

    Wang, Hui; Yang, Lei; Deng, Jieqiong; Wang, Bo; Yang, Xiaorong; Yang, Rongrong; Cheng, Mei; Fang, Wenxiang; Qiu, Fuman; Zhang, Xin; Ji, Weidong; Ran, Pixin; Zhou, Yifeng; Lu, Jiachun

    2014-09-01

    Lung inflammation and epithelial to mesenchymal transition (EMT) are two pathogenic features for the two contextual diseases: chronic obstructive pulmonary disease (COPD) and lung cancer. VEGFR1 (or FLT1) plays a certain role in promoting tumour growth, inflammation and EMT. To simultaneously test the association between the single nucleotide polymorphisms (SNPs) in VEGFR1 and risk of COPD and lung cancer would reveal genetic mechanisms shared by these two diseases and joint aetiology. We conducted a two-population hospital-based case-control study. Three potential functional SNPs (rs664393, rs7326277 and rs9554314) were genotyped in southern Chinese and validated in eastern Chinese to explore their associations with COPD risk in 1511 COPD patients and 1677 normal lung function controls, and with lung cancer risk in 1559 lung cancer cases and 1679 cancer-free controls. We also detected the function of the promising SNP. Individuals carrying the rs7326277C (CT+CC) variant genotypes of VEGFR1 had a significant decrease in risk of both COPD (OR = 0.78; 95% CI = 0.68-0.90) and lung cancer (OR = 0.79; 95% CI = 0.64-0.98), compared with those carrying the rs7326277TT genotype. Functional assays further showed that the rs7326277C genotypes had lower transcriptional activity and caused decreased VEGFR expression, compared with the rs7326277TT genotype. However, no significant association was observed for the other two SNPs (rs664393 and rs9554314) and either COPD or lung cancer risk. Our data suggested that the rs7326277C variant of VEGFR1 could reduce both COPD and lung cancer risk by lowering VEGFR1 mRNA expression; the SNP might be a common susceptible locus for both COPD and lung cancer.

  4. rs10719 Polymorphism Located within DROSHA 3′-Untranslated Region is Responsible for Development of Primary Hypertension by Disrupting Binding with microRNA-27b

    PubMed Central

    Zhang, Yabing; Cao, Ai-lin; Dong, Chun

    2017-01-01

    Background MiR-27b is reportedly involved with many diseases (e.g., gastric cancer) by acting on different signaling pathways. In this study, we aimed at understanding the relationship between miR-27b and hypertension and its underlying molecular mechanism. Material/Methods Peripheral blood was collected from patients with hypertension, and statistical analysis was performed to study the association between rs10719 and risk of hypertension. Tissue samples were collected from patients with lung cancer, and the expression of miR-27b and DROSHA was determined using Western blot analysis and real-time PCR. Results We first searched the miRNA database online, and identified DROSHA as a virtual target of miR-27b with the “seed sequence” located within the 3′-UTR of the target gene, and then validated DROSHA to be the direct gene via luciferase reporter assay system. We also established the negative regulatory relationship between miR-27b and DROSHA via studying the relative luciferase activity. We also conducted real-time PCR to study the mRNA and protein expression level of miR-27b among different groups. Furthermore, we conducted real-time PCR and densitometry analysis to study the mRNA and protein expression level of DROSHA among different groups of cells treated with scramble control, miR-27b mimics, DROSHA siRNA, and miR-27b inhibitors to verify the negative regulatory relationship between MiR-27b and DROSHA. Conclusions The presence of rs10719 disrupted the interaction between miR-27b and DROSHA, which might be the underlying mechanism of the observation that rs10719 is significantly associated with risk of primary hypertension. PMID:28214904

  5. Loss of estrogen receptor beta isoform expression and its correlation with aberrant DNA methylation of the 5'-untranslated region in human epithelial ovarian carcinoma.

    PubMed

    Suzuki, Fumihiko; Akahira, Jun-Ichi; Miura, Ikumi; Suzuki, Takashi; Ito, Kiyoshi; Hayashi, Shin-Ichi; Sasano, Hironobu; Yaegashi, Nobuo

    2008-12-01

    Evidence exists that sex steroids such as estrogens affect epithelial ovarian cancer. The expression profiles of the estrogen receptors (ER) and ERbeta in particular have not been fully described. Therefore, in our present study, we examined the methylation status of the promoters 0K and 0N, and the expression of ERbeta isoforms in human epithelial ovarian carcinoma. We then correlated methylation status with ER expression status. Twelve ovarian carcinoma cell lines, six primary cultures of ovarian surface epithelial cells (OSE), and 64 cases of ovarian carcinoma tissues were examined. Bisulfite sequencing and quantitative reverse transcription-polymerase chain reaction were used to evaluate methylation status and expression of ERbeta isoforms. The relative abundance of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA was significantly lower in ovarian cancer cell lines and tissues than in their corresponding normal counterparts. However, ERbeta5 mRNA level was relatively higher in the cancers, in clear cell adenocarcinoma in particular, than in the normal ovary. Bisulfite sequencing analysis demonstrated that the two promoters of the ERbeta gene exhibited distinct methylation patterns. Promoter 0N was unmethylated in OSE, rarely methylated in normal ovarian tissues, and extensively methylated in ovarian cancer cell lines and tissues (11/15 cell lines and 18/32 cancer tissues were extensively methylated). The promoter 0K was, however, unmethylated in both normal and malignant ovarian cells and tissues. A significant correlation between promoter 0N hypermethylation and the loss of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA expression was detected in ovarian carcinoma cells and tissues. Treatment of ovarian carcinoma cells with 5-aza-2' deoxycytidine resulted in reexpression of the ERbeta gene. The results of our present study suggest that ERbeta is inactivated mainly through aberrant DNA methylation. This process may play an important role in the pathogenesis of epithelial ovarian cancer.

  6. A single-nucleotide polymorphism in the 3' untranslated region of the LPIN1 gene and association analysis with performance traits in chicken.

    PubMed

    Zhang, S P; Li, S Y; Chen, W; Lu, W W; Huang, Y Q

    2013-06-01

    1. A single nucleotide polymorphism (SNP), c.*77C>G, was found in the 3' UTR of the chicken LPIN1 gene by DNA sequencing. In total, 860 chickens were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a F2 resource population obtained by crossing F0 Gushi chickens and Anka broilers, and the associations of this polymorphism with chicken growth, carcass, muscle fibre traits and serum biochemistry parameters were analysed. 2. Significant associations were found between the polymorphism and breast muscle fibre diameter (FDB). Comparison of the different genotypes of c.*77C>G in the F2 resource population showed that the GG genotype had significantly higher values than that of CG genotype in FDB. c.*77C>G was predicted to cause changes to multiple microRNA (miRNA) binding sites. But the total mRNA level of chicken LPIN1, LPIN1-;α and LPIN1-β in liver and muscle tissues did not show significant difference among GG, CG and CC genotypes, respectively. 3. The results suggested that chicken LPIN1 has a potential effect on muscle fibre development, but no effect on other studied traits.

  7. Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5' untranslated region of hepatitis C virus RNA.

    PubMed

    Biegel, Jason M; Henderson, Eric; Cox, Erica M; Bonenfant, Gaston; Netzband, Rachel; Kahn, Samantha; Eager, Rachel; Pager, Cara T

    2017-07-01

    Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5' UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. HLA-E coding and 3' untranslated region variability determined by next-generation sequencing in two West-African population samples.

    PubMed

    Castelli, Erick C; Mendes-Junior, Celso T; Sabbagh, Audrey; Porto, Iane O P; Garcia, André; Ramalho, Jaqueline; Lima, Thálitta H A; Massaro, Juliana D; Dias, Fabrício C; Collares, Cristhianna V A; Jamonneau, Vincent; Bucheton, Bruno; Camara, Mamadou; Donadi, Eduardo A

    2015-12-01

    HLA-E is a non-classical Human Leucocyte Antigen class I gene with immunomodulatory properties. Whereas HLA-E expression usually occurs at low levels, it is widely distributed amongst human tissues, has the ability to bind self and non-self antigens and to interact with NK cells and T lymphocytes, being important for immunosurveillance and also for fighting against infections. HLA-E is usually the most conserved locus among all class I genes. However, most of the previous studies evaluating HLA-E variability sequenced only a few exons or genotyped known polymorphisms. Here we report a strategy to evaluate HLA-E variability by next-generation sequencing (NGS) that might be used to other HLA loci and present the HLA-E haplotype diversity considering the segment encoding the entire HLA-E mRNA (including 5'UTR, introns and the 3'UTR) in two African population samples, Susu from Guinea-Conakry and Lobi from Burkina Faso. Our results indicate that (a) the HLA-E gene is indeed conserved, encoding mainly two different protein molecules; (b) Africans do present several unknown HLA-E alleles presenting synonymous mutations; (c) the HLA-E 3'UTR is quite polymorphic and (d) haplotypes in the HLA-E 3'UTR are in close association with HLA-E coding alleles. NGS has proved to be an important tool on data generation for future studies evaluating variability in non-classical MHC genes.

  9. The 3' untranslated region of the two cytosolic glutamine synthetase (GS(1)) genes in alfalfa (Medicago sativa) regulates transcript stability in response to glutamine.

    PubMed

    Simon, Bindu; Sengupta-Gopalan, Champa

    2010-10-01

    Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to produce glutamine. The GS enzyme is located either in the chloroplast (GS(2)) or in the cytoplasm (GS(1)). GS(1) is encoded by a small gene family and the members exhibit differential expression pattern mostly attributed to transcriptional regulation. Based on our recent finding that a soybean GS(1) gene, Gmglnβ ( 1 ) is subject to its 3'UTR-mediated post-transcriptional regulation as a transgene in alfalfa (Medicago sativa) we have raised the question of whether the 3'UTR-mediated transcript destabilization is a more universal phenomenon. Gene constructs consisting of the CaMV35S promoter driving the reporter gene, GUS, followed by the 3'UTRs of the two alfalfa GS(1) genes, MsGSa and MsGSb, were introduced into alfalfa and tobacco. The analysis of these transformants suggests that while both the 3'UTRs promote transcript turnover, the MsGSb 3'UTR is more effective than the MsGSa 3'UTR. However, both the 3'UTRs along with Gmglnβ ( 1 ) 3'UTR respond to nitrate as a trigger in transcript turnover. More detailed analysis points to glutamine rather than nitrate as the mediator of transcript turnover. Our data suggests that the 3'UTR-mediated regulation of GS(1) genes at the level of transcript turnover is probably universal and is used for fine-tuning the expression in keeping with the availability of the substrates.

  10. RNA-Mediated Virus Resistance: Role of Repeated Transgenes and Delineation of Targeted Regions.

    PubMed Central

    Sijen, T.; Wellink, J.; Hiriart, J. B.; Van Kammen, A.

    1996-01-01

    Resistance to cowpea mosaic virus (CPMV) in transgenic Nicotiana benthamiana plants is RNA mediated. In resistant CPMV movement protein (MP) gene-transformed lines, transgene steady state mRNA levels were low, whereas nuclear transcription rates were high, implying that a post-transcriptional gene-silencing mechanism is at the base of the resistance. The silencing mechanism can also affect potato virus X (PVX) RNAs when they contain CPMV MP gene sequences. In particular, sequences situated in the 3[prime] part of the transcribed region of the MP transgene direct elimination of recombinant PVX genomes. Remarkably, successive portions of this 3[prime] part, which can be as small as 60 nucleotides, all tag PVX genomes for degradation. These observations suggest that the entire 3[prime] part of the MP transgene mRNA is the initial target of the silencing mechanism. The arrangement of transgenes in the plant genome plays an important role in establishing resistance because the frequency of resistant lines increased from 20 to 60% when transformed with a transgene containing a direct repeat of MP sequences rather than a single MP transgene. Interestingly, we detected strong methylation in all of the plants containing directly repeated MP sequences. In sensitive lines, only the promoter region was found to be heavily methylated, whereas in resistant lines, only the transcribed region was strongly methylated. PMID:12239378

  11. Synthesis of 25-hydroxyvitamin D sub 3 3. beta. -3 prime -(N-(4-azido-2-nitrophenyl)amino)propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D sub 3 : Photoaffinity labeling of rat serum vitamin D binding protein

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Van Baelen, H. )

    1991-05-14

    Vulnerability of 25-hydroxy-(26,27-{sup 3}H)vitamin D{sub 3} 3{beta}-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D{sub 3} (25-OH-D{sub 3}) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D{sub 3}, and 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D{sub 3} binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with {sup 3}H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D{sub 3}. These results provide strong evidence for the covalent labeling of the 25-OH-D{sub 3} binding site in rDPB by {sup 3}H-25-ANE.

  12. Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

    PubMed Central

    1986-01-01

    Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase. PMID:3536960

  13. Linkage disequilibrium in the neurofibromatosis I (NF1) region: Implications for gene mapping

    SciTech Connect

    Jorde, L.B.; Watkins, W.S.; Viskochil, D.; Ward, K. ); O'Connell, P. )

    1993-11-01

    To test the usefulness of linkage disequilibrium for gene mapping, the authors compared physical distances and linkage disequilibrium among eight RFLPs in the neutrofibromatosis 1 (NF1) region. Seven of the polymorphisms span most of the NF1 gene, while the remaining polymorphism lies approximately 70 kb 3[prime] to a stop codon in exon 49. By using Centre d'Etude du Polymorphisme Humain (CEPH) kindreds, 91-110 unrelated parents were genotyped. A high degree of disequilibrium is maintained among the seven intragenic polymorphisms (r > .82, P < 10[sup [minus]7]), even though they are separated by as much as 340 kb. The 3[prime] polymorphism is only 68 kb distal to the next polymorphism, but disequilibrium between the 3[prime] polymorphism and all others is comparatively low ([vert bar]r[vert bar] < .33, P values .27-.001). This result was replicated in three sets of unrelated kindreds: the Utah CEPH families, the non-Utah CEPH families, and an independent set of NF1 families. Trigenic, quadrigenic, three-locus, and four-locus disequilibrium measures were also estimated. There was little evidence of higher-order linkage disequilibrium. As expected for a disease with multiple mutations, no disequilibrium was observed between the disease gene and any of the RFLPs. The observed pattern of high disequilibrium within the gene and a loss of disequilibrium 3[prime] to the stop codon could have implications for gene mapping studies. These are discussed, and guidelines for linkage disequilibrium studies are suggested. 80 refs., 2 figs., 5 tabs.

  14. Difference in hepatic metallothionein content in Antarctic red-blooded and haemoglobinless fish: undetectable metallothionein levels in haemoglobinless fish is accompanied by accumulation of untranslated metallothionein mRNA.

    PubMed Central

    Scudiero, R; Carginale, V; Riggio, M; Capasso, C; Capasso, A; Kille, P; di Prisco, G; Parisi, E

    1997-01-01

    Icefish (family Channichthyidae, suborder Nothothenioidei) are a group of Antarctic fish that have evolved unique phenotypes in order to adapt to the environment in which they live. Besides the lack of haemoglobin and the drastic reduction in the number of erythrocyte-like cells, another striking feature of the icefish is that their liver is devoid of metallothionein. These cysteine-rich heavy-metal-binding proteins are usually present in large amounts in a large variety of organisms, from bacteria to mammals. Despite the failure to detect appreciable levels of metallothionein in icefish liver, a cDNA encoding metallothionein was produced from total RNA by reverse transcriptase PCR. The icefish metallothionein showed high percentage identity with metallothionein from Trematomus bernachii, a red-blooded Antarctic fish in which a normal content of hepatic metallothionein was found. Steady-state mRNA levels were assessed in fish liver by high-stringency hybridization of the metallothionein probe with total RNA. The results showed that icefish livers retain large amounts of untranslated metallothionein mRNA. The stability of the icefish transcript might be correlated with the lack of specific motifs in the untranslated 3' ends of mRNA. PMID:9078263

  15. Effects of 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or an extract derived from field-collected cormorant eggs injected into double-crested cormorant (Phalacrocorax auritus) eggs

    SciTech Connect

    Powell, D.C.; Aulerich, R.J.; Powell, J.F.; Restum, J.C.; Giesy, J.P.; Bursian, S.J.; Meadows, J.C.; Tillitt, D.E.; Stromborg, K.L.

    1997-07-01

    Double-crested cormorant (Phalacrocorax auritus) eggs were injected with either 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or an extract derived from field-collected double-crested cormorant eggs. These compounds were injected into the yolks of cormorant eggs from an isolated colony on Lake Winnipegosis, Manitoba, Canada. Upon hatching, chicks were necropsied. The brain, bursa, heart, liver, and spleen were removed and weighed. An approximate median lethal dose (LD50) of 158 {micro}g/kg egg was determined for PCB 126, which is 69 times greater than the LD50 determined for the chicken (Gallus domesticus) in a previous study. A significantly greater mortality occurred at the highest dose of TCDD when compared to the vehicle control. However, the mortality data did not provide sufficient information for the determination of an LD50. The cormorant egg extract did not adversely affect hatchability. No significant increases were observed in the incidence of developmental abnormalities, including pronounced edema, in any of the treatment groups, nor were there any relevant effects on body and organ weights. Based on the results from this study, the cormorant appears to be considerably less sensitive to polyhalogenated diaromatic hydrocarbons than the chicken, which has been the typical species used for egg injection studies.

  16. Interleukin-3 priming in acute myeloid leukaemia patients.

    PubMed

    Tafuri, A; de Felice, L; Goodacre, A; Fenu, S; Petrucci, M T; Valentini, T; Alimena, G; Petti, M C; Meloni, G; Mandelli, F

    1995-09-01

    Several studies have demonstrated that G-CSF, GM-CSF and, in particular, IL-3 can effectively recruit acute myeloid leukaemia (AML) blasts into the cell cycle, resulting in a significant increase in cytosine-arabinoside (Ara-C) mediated cytotoxicity in vitro. Since IL-3 has shown biological and clinical activity, we investigated the cell kinetic effects of rIL-3 and high-dose Ara-C/idarubicin in three patients with refractory AML selected for the presence of chromosome 7 monosomy; this enabled differentiation between the effects of IL-3 on leukaemic and on normal cells. The in vivo administration of rhIL-3 (250 micrograms/m2d s.c. for 6-10d) recruited AML blasts into the cell cycle in two of the three patients, and this effect resulted in an increase in in vitro growth of clonogenic cells (CFU-L) and of their S-phase fraction. The percentage of leukaemic cells with monosomy 7 increased only in the two cases who showed a proliferative response. Normal cells were not recruited, even when rhIL-3 was administered for up to 10 d. In vitro studies showed an increased Ara-C cytotoxicity on clonogenic AML cells, in particular with IL-3 plus GM-CSF, thus confirming the priming effects of IL-3 in the two responding cases. The results of this study suggest that rhIL-3 can selectively recruit leukaemic cells into the cell cycle. Although leukaemic blasts can be sensitized to Ara-C, other mechanisms of primary blast resistance may limit the clinical benefit of kinetic-based approaches.

  17. Higher order structures of the 5'-proximal region decrease the efficiency of translation of the porcine pro-opiomelanocortin mRNA.

    PubMed

    Chevrier, D; Vézina, C; Bastille, J; Linard, C; Sonenberg, N; Boileau, G

    1988-01-15

    The SP6 polymerase/promoter system was used to synthesize porcine pro-opiomelanocortin mRNAs with nucleotide sequence deletions in the 5'- as well as 3'-untranslated and coding regions. The translational efficiency of the mutant mRNAs was evaluated by cell-free translation or by monitoring the rate and extent of ribosome binding in the presence of sparsomycin. The results of these experiments indicate that specific nucleotide sequences in the 5'-untranslated and coding regions of the pro-opiomelanocortin mRNA decrease its rate of translation. Structure mapping of the mRNA with double-strand and single-strand specific nucleases suggests that these sequences can form stable secondary structures.

  18. SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

    PubMed Central

    Mix, Heiko; Lobanov, Alexey V.; Gladyshev, Vadim N.

    2007-01-01

    Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3′-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins. PMID:17169995

  19. An untranslated insertion variant in the uncoupling protein 2 gene is not related to body mass index and changes in body weight during a 26-year follow-up in Danish Caucasian men.

    PubMed

    Dalgaard, L T; Sørensen, T I; Andersen, T; Hansen, T; Pedersen, O

    1999-12-01

    Associations between a 45 bp 3'untranslated insertion polymorphism in the uncoupling protein 2 (UCP2) gene and both body mass index (BMI) and sleeping metabolic rate have previously been reported. We investigated the impact of this polymorphism on BMI and long-term body weight changes. The allelic frequency of the UCP2 insertion variant was determined in a cohort of 744 obese Danish Caucasian men who had a BMI of at least 31 kg/m2 at the draft-board examinations and a randomly selected control cohort consisting of 872 draftees. Follow-up measurements of BMI were done on average 26 years after the draft-board examinations. The prevalence of the insertion allele was 30.4% (95% confidence interval: 28.0-32.8%) among the obese and 29.6% (27.4-31.8%) in the control group (p = 0.6). In a lean group selected as the 354 subjects with a BMI less than 25 kg/m2 at 46 years of age from the control group, the frequency of insertion allele was 29.0% (27.2-30.8%) (p = 0.5 compared with the obese cohort). The BMI at the ages of 20 and 46 years did not differ between genotypes either in the obese or the control group. Similarly, the changes in BMI/year between examinations at 20 and 46 years of age did not differ between genotypes in either group. In a large group of Danish Caucasian men we found no association between a 3'untranslated insertion polymorphism in the UCP2 gene and obesity. Neither did we identify a relation between this variant and BMI changes during adult age.

  20. High-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the entire human DiGeorge syndrome critical region 2 (DGCR2) gene at 22q11.2.

    PubMed

    Iida, A; Ohnishi, Y; Ozaki, K; Ariji, Y; Nakamura, Y; Tanaka, T

    2001-01-01

    We constructed a high-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the DiGeorge syndrome critical region 2 (DGCR2) gene at chromosome 22q11.2, a human counterpart of mouse seizure-related gene SEZ-12. A total of 102 SNPs were isolated from the region by systematic screening among 48 Japanese individuals: 9 SNPs in the 5' flanking region, 3 in the 5' untranslated region, 2 in the coding regions, 77 in introns, 7 in the 3' untranslated region, and 4 in the 3' flanking region. By a comparison of our data with SNPs deposited in the dbSNP database in the National Center for Biotechnology Information, 80 SNPs (78.4%) were considered to be novel. The ratio of transition to transversion was 3.08:1. In addition, eight other types of genetic variations (one GA dinucleotide polymorphism and seven insertion/deletion polymorphisms) were discovered. The high-resolution map that we constructed will be a useful resource for analyzing gene scans of complex diseases mapped to this local segment on chromosome 22.

  1. Analysis of the genetic diversity of the Plasmodium falciparum multidrug resistance gene 5' upstream region.

    PubMed

    Myrick, Alissa; Sarr, Ousmane; Dieng, Therese; Ndir, Omar; Mboup, Souleymane; Wirth, Dyann F

    2005-02-01

    Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal. A comparison of six geographically diverse laboratory strains showed that the 1.94-kb 5'-untranslated region is highly monomorphic, with a total of four unique single nucleotide polymorphisms (SNPs) being identified. All of the mutations were localized to a 462-basepair region proximal to the transcription start point. Analysis of this region in field isolates shows the prevalence of one SNP throughout the entire population of parasites, irrespective of drug resistance status. The SNP frequency of the pfmdr1 upstream region is lower than that found in the noncoding region of other genes.

  2. Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain.

    PubMed Central

    Sasso, M P; Carsana, A; Confalone, E; Cosi, C; Sorrentino, S; Viola, M; Palmieri, M; Russo, E; Furia, A

    1991-01-01

    In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined. Images PMID:1754384

  3. The up regulation of phosphofructokinase1 (PFK1) protein during chemically induced hypoxia is mediated by the hypoxia-responsive internal ribosome entry site (IRES) element, present in its 5'untranslated region.

    PubMed

    Ismail, Rehana; Ul Hussain, Mahboob

    2017-08-01

    Astrocytes cope-up the hypoxia conditions by up regulating the activity of the enzymes catalyzing the irreversible steps of the glycolytic pathway. The phosphofructokinase1 (PFK1), which converts fructose-6-phosphate to fructose-1, 6-bisphosphate, is the major regulatory enzyme of the glycolytic pathway. For this purpose, we investigated the expression regulation of the PFK1 during chemically induced hypoxia. After 48 h of the chemically induced hypoxia induction of the C6 glioma cells, the PFK1 protein depicted strong up regulation, with no appreciable change in its mRNA levels. The di-cistronic assay indicated the presence of a weak internal ribosome entry site (IRES) element in the 5'UTR of the PFK1 mRNA. Interestingly, the weak IRES element of the PFK1 was strongly up regulated after 48 h of the chemically induced hypoxia, indicative of a possible mechanism responsible for the induction of the PFK1 protein. The authenticity of the hypoxia-regulated IRES element of the PFK1, relative to the presence of the cryptic promoter element and/or the cryptic splicing was established using promoterless di-cistronic assay and the RT-PCR analysis. Moreover, the ectopic expression of the polypyrimidine tract binding (PTB) protein resulted in the enhanced activity of the IRES element of the PFK1. Additionally, it was established that the chemically induced hypoxia resulted in the increased shuttling of the PTB from the cell nucleus to the cytosol. The presence of a hypoxia responsive IRES element, in the 5'UTR of the PFK1 was established to be the possible mechanism responsible for the up regulation of the PFK1 protein. Our data provides an interesting mechanism that may explain the increased glycolytic capacity of the astrocytes after brain hypoxia. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  4. The turnip yellow mosaic virus tRNA-like structure cannot be replaced by generic tRNA-like elements or by heterologous 3' untranslated regions known to enhance mRNA expression and stability.

    PubMed Central

    Skuzeski, J M; Bozarth, C S; Dreher, T W

    1996-01-01

    The tRNA-like structure (TLS) at the 3' end of the turnip yellow mosaic virus genome was replaced with heterologous tRNA-like elements, and with a poly(A) tail, in order to assess its role. Replacement with the valylatable TLSs from two closely related tymoviruses resulted in infectious viruses. In contrast, no systemic symptoms on plants, and only low viral accumulations in protoplasts, were observed for three chimeric genomes with 3' sequences known to enhance mRNA stability and translatability. One of these chimeras had a poly(A) tail, and the others had the TLS with associated upstream pseudoknot tracts from the 3' ends of brome mosaic and tobacco mosaic viruses. The latter two chimeric RNAs were shown to be appropriately folded by demonstrating their aminoacylation in vitro with tyrosine and histidine, respectively. The results show that enhancement of genome stability or gene expression is not the major role of the turnip yellow mosaic virus TLS. The major role is likely to be replicational, dependent on features present in tymoviral TLSs but not in generic tRNA-like structures. PMID:8642631

  5. A SNP in 5′ untranslated region of CD40 gene is associated with an increased risk of ischemic stroke in a Chinese population: a case-control study

    PubMed Central

    Huang, Hua-Tuo; Guo, Jing; Xiang, Yang; Chen, Jian-Ming; Luo, Hong-Cheng; Meng, Lan-Qing; Wei, Ye-Sheng

    2017-01-01

    Abstract Cluster of differentiation 40 (CD40), the receptor for CD154, is a member of the tumor necrosis factor (TNF) receptor superfamily. Several studies have been conducted to investigate the effect of the CD40 rs1883832 polymorphism on atherosclerotic disease in different population; however, inconsistent results were obtained. In this study, we investigated the association of four polymorphisms (rs1883832, rs13040307, rs752118 and rs3765459) of CD40 gene and their effect on CD40 expression with the risk of ischemic stroke (IS) in a Chinese population. Three hundred and eighty patients with IS and 450 control subjects were included in the study. The CD40 polymorphisms were discriminated by Snapshot SNP genotyping assay. Serum soluble CD40 (sCD40) levels were detected by ELISA. We found that the rs1883832CT and rs1883832TT genotypes were associated with an increased risk of IS compared with the rs1883832CC genotype (OR = 1.42, 95% CI: 1.03–1.95, p = 0.030 and OR = 1.91, 95% CI: 1.29–2.82, P = 0.001, respectively), and the rs1883832T allele was associated with a significantly increased risk of IS compared with rs1883832C allele (OR = 1.40, 95% CI: 1.15–1.70, P = 0.001). Elevated serum sCD40 levels were observed in patients with IS compared with the control gropu (P < 0.01). Individuals carrying the rs1883832TT or rs1883832CT genotypes showed significantly higher sCD40 levels compared with the rs1883832CC genotype in the IS group [(64.8 ± 25.4 pg/mL, TT = 94); (63.9 ± 24.3 pg/mL, CT = 185) vs (53.3 ± 22.5 pg/mL, CC = 101), P < 0.01]. The TCCA haplotype was associated with an increased risk of IS compared with the control group (OR = 2.10, 95% CI: 1.23–3.58, p = 0.005). However, we did not find a significant association between the other three polymorphisms and IS risk. In conclusion, after a comprehensive comparison with other studies, we confirmed that the rs1883832T allele but not the rs1883832C allele is associated with an increased risk of IS. The rs1883832 polymorphism may exert influences on abnormal CD40 expression in IS patients among the Chinese population. PMID:28590502

  6. Mutation creates an open reading frame within the 5' untranslated region of macaque erythrocyte carbonic anhydrase (CA) I mRNA that suppresses CA I expression and supports the scanning model for translation.

    PubMed Central

    Bergenhem, N C; Venta, P J; Hopkins, P J; Kim, H J; Tashian, R E

    1992-01-01

    A variant allele at the CA I locus that produces a deficiency of erythrocyte-specific CA I occurs as a widespread polymorphism in pigtail macaques from southeast Asia. Sequence analyses revealed a C----G substitution 12 nucleotides downstream of the cap site in the variant erythrocyte CA I mRNA. This mutation forms a new AUG start site and an open reading frame coding for 26 amino acids that terminates 6 nucleotides before the normal AUG initiation codon for CA I. It appears that the presence of this upstream open reading frame greatly diminishes reinitiation of translation from the normal start site, resulting in trace levels of CA I in erythrocytes. Preferential use of the first AUG codon supports the scanning model for translation initiation in eukaryotes. Images PMID:1528895

  7. Analysis of the 3' untranslated regions of alpha-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas.

    PubMed

    Kelly, Shannan; Yamamoto, Hideki; Robles, Laura J

    2008-08-04

    We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)-dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in alpha-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. 3'-RACE and sequencing were used to isolate and analyze the 3'-UTRs of alpha-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. The following CPE-like sequence was detected in the 3'-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3'-UTRs of alpha-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3'-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of alpha-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas.

  8. Analysis of the 3’ untranslated regions of α-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas

    PubMed Central

    Kelly, Shannan; Yamamoto, Hideki

    2008-01-01

    Purpose We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)–dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in α-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. Methods 3’-RACE and sequencing were used to isolate and analyze the 3’-UTRs of α-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. Results The following CPE-like sequence was detected in the 3’-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3’-UTRs of α-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. Conclusions The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3’-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of α-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas. PMID:18682811

  9. Naturally occurring human genetic variation in the 3’-untranslated region of the secretory protein chromogranin A (CHGA) is associated with autonomic blood pressure regulation and hypertension in sex-dependent fashion

    PubMed Central

    Chen, Yuqing; Rao, Fangwen; Rodriguez-Flores, Juan L.; Mahata, Manjula; Fung, Maple M.; Stridsberg, Mats; Vaingankar, Sucheta M.; Wen, Gen; Salem, Rany M.; Das, Madhusudan; Cockburn, Myles G.; Schork, Nicholas J.; Ziegler, Michael G.; Hamilton, Bruce A.; Mahata, Sushil K.; Taupenot, Laurent; O’Connor, Daniel T.

    2008-01-01

    Objective Determination whether common variation at the CHGA locus increases susceptibility to hypertension. Background Chromogranin A (CHGA) regulates catecholamine storage and release. Previously we systematically identified genetic variants across CHGA. Methods Dense genotyping across the CHGA locus in >1000 individuals with the most extreme BPs in the population, as well twin pairs with autonomic phenotypes. Characterizing function of a trait-associated 3'-UTR variant with transfected CHGA 3'-UTR/luciferase reporter plasmids. Results CHGA was overexpressed in patients with hypertension, especially hypertensive men, and CHGA predicted catecholamines. In individuals with extreme BPs, CHGA genetic variants predicted BP, especially in men, with a peak association occurred in the 3'-UTR at C+87T, accounting for up to ~12/~9 mmHg. The C+87T genotype predicted CHGA secretion in vivo, with the +87T allele (associated with lower BP) also diminishing plasma CHGA by ~10%. The C+87T 3'-UTR variant also predicted the BP response to environmental (cold) stress; the same allele (+87T) that diminished basal BP in the population also decreased the SBP response to stress by ~12 mmHg, and the response was smaller in women (by ~6 mmHg). In a chromaffin cell-transfected CHGA 3'-UTR/luciferase reporter plasmid, the +87T allele associated with lower BP also decreased reporter expression by ~30%. In cultured chromaffin cells, reducing endogenous Chga expression by si-RNA caused ~2/3 depletion of catecholamine storage vesicles. Conclusions Common variant C+87T in the CHGA 3'-UTR is a functional polymorphism causally associated with hypertension especially in men of the population, and propose steps ("intermediate phenotypes") whereby in sex-dependent fashion this genetic variant influences the ultimate disease trait. These observations suggest new molecular strategies to probe the pathophysiology, risk, and rational treatment of hypertension. PMID:19017515

  10. Introduction of mutations into the non-structural genes or 3' untranslated region of an attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity.

    PubMed

    Hanley, Kathryn A; Manlucu, Luella R; Manipon, Gracielle G; Hanson, Christopher T; Whitehead, Stephen S; Murphy, Brian R; Blaney, Joseph E

    2004-09-03

    A dengue virus vaccine candidate, rDEN4Delta30, has been previously reported to be safe and immunogenic in humans, but a subset of vaccinees developed asymptomatic rash, elevation of liver enzymes and/or mild neutropenia. In the current study, mutations that had previously been shown to reduce replication of DEN4 virus in suckling mice and/or in SCID mice engrafted with human liver cells (SCID-HuH-7 mice) were introduced into rDEN4Delta30 in an attempt to further attenuate this virus. Three of the five resulting modified rDEN4Delta30 viruses showed decreased replication in SCID-HuH-7 mice relative to rDEN4Delta30. Moreover, in rhesus monkeys, two of the modified rDEN4Delta30 viruses showed a decrease in replication relative to rDEN4Delta30 while generating levels of neutralizing antibody similar to rDEN4Delta30 virus. All of the modified rDEN4Delta30 viruses completely protected immunized rhesus monkeys from challenge with wild-type DEN4 virus. Based on their attenuation for both human liver cells and rhesus monkeys, two of the modified rDEN4Delta30 vaccine candidates are currently being prepared for use in clinical trials. The application of these attenuating mutations to flavivirus vaccine development is discussed.

  11. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae

    SciTech Connect

    Stewart, P.; Whitwam, R.E.; Tien, Ming

    1996-03-01

    A manganese peroxidase (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3{prime} untranslated region of the glucoamylase gene of Asperigillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. 34 refs., 7 figs., 1 tab.

  12. Genomic organization of the ATM gene

    SciTech Connect

    Uziel, T.; Savitsky, K.; Platzer, M.; Rosenthal, A.

    1996-04-15

    The ATM gene was recently identified and found to be responsible for the human genetic disorder ataxia-telangiectasia. The major ATM transcript is 13 kb. Using long-distance PCR, we determined the genomic structure of this gene and identified all of its exon-intron boundaries. The ATM gene spans approximately 150 kb of genomic DNA and consists of 66 exons. The initiation codon falls within exon 4. The last exon is 3.8 kb and contains the stop codon and a 3{prime}-untranslated region of about 3600 nucleotides. 19 refs., 2 figs., 1 tab.

  13. Region between the canine distemper virus M and F genes modulates virulence by controlling fusion protein expression.

    PubMed

    Anderson, Danielle E; von Messling, Veronika

    2008-11-01

    Morbilliviruses, including measles and canine distemper virus (CDV), are nonsegmented, negative-stranded RNA viruses that cause severe diseases in humans and animals. The transcriptional units in their genomes are separated by untranslated regions (UTRs), which contain essential transcription and translation signals. Due to its increased length, the region between the matrix (M) protein and fusion (F) protein open reading frames is of particular interest. In measles virus, the entire F 5' region is untranslated, while several start codons are found in most other morbilliviruses, resulting in a long F protein signal peptide (Fsp). To characterize the role of this region in morbillivirus pathogenesis, we constructed recombinant CDVs, in which either the M-F UTR was replaced with that between the nucleocapsid (N) and phosphoprotein (P) genes, or 106 Fsp residues were deleted. The Fsp deletion alone had no effect in vitro and in vivo. In contrast, substitution of the UTR was associated with a slight increase in F gene and protein expression. Animals infected with this virus either recovered completely or experienced prolonged disease and death due to neuroinvasion. The combination of both changes resulted in a virus with strongly increased F gene and protein expression and complete attenuation. Taken together, our results provide evidence that the region between the morbillivirus M and F genes modulates virulence through transcriptional control of the F gene expression.

  14. BCL2 major breakpoint region (mbr) may specify an origin of replication

    SciTech Connect

    DiCroce, P.A.; Bailey, S.; Hagins, W.C.

    1994-09-01

    We have described a minisatellite consensus signal, GC[A/T]GG[A/T]GG, which resembles the prokaryotic activator of recombination, {chi}. The consensus appears frequently at the breakpoints of oncogene translocations, especially those in which the V(D)J recombinase has been implicated. We have investigated this relationship by examining the breakpoint positions and DNA sequence of many mbr translations from human follicular lymphomas. Breakpoints occur in three, evenly-spaced clusters 14-18 bp wide and 50 bp apart; the first cluster begins at the first base 3{prime} to the {chi} signal. At the end of cluster 3, translocations abruptly decline in frequency. We now report that this region is characterized by multiple binding sites for both single- and double-strand DNA binding proteins. Furthermore, the binding sites immediately flank clusters 1 and 3, thus defining the region at risk for translocation. The two single-strand binding proteins, one each for the sense and anti-sense strand of BCL2, bind the {chi} signal that marks the onset of translocation. The second binding site, which begins at the 3{prime} flank of cluster 3, extends a further 80 bp downstream and is absolutely required for the interaction of the mbr with factor(s) which can denature the target DNA in a cell-specific fashion. This process requires energy, as complex formation is inhibited by ATP{gamma}S. One of the two ssDNA binding proteins and, possibly, the helicase activity are expressed in a cell-cycle-dependent fashion. Finally, helical stability studies of the helicase binding region yield a profile comparable to those previously defined for several viral, yeast, and bacterial origins of replication. These studies indicate that BCL2 translocation may reflect (1) a requirement for removing a replication origin from the gene to promote lymphomagenesis and/or (2) the recombinogenic nature of such structures.

  15. Gene search in the FSHD region on 4q35

    SciTech Connect

    Deutekom, J.C.T. van; Romberg, S.; Geel, M. van

    1994-09-01

    In the search for the FSHD gene on 4q35, four overlapping cosmids spanning a region of 95 kb including the deletion-prone repeated units were subcloned as well as subjected to cDNA selection and exon trap strategies. A total of 300 selected clones with an average length of 500 bp were mapped back to the cosmids. None of the clones appeared to be single copy. Sequence data of most clones and the related genomic regions were compared. cDNA clones with a high homolgy (>90%) and a low repetitive hybridization pattern were further analyzed by Zoo- and Northern blotting and by sequence analysis programs like GRAIL. Excellent and good exons could be identified and some clones showed evolutionary conservation. With the best cDNA, genomic and exon trap clones, several cDNA libraries were screened. The obtained cDNAs identified different genes, none of which originated from 4q35. 3{prime} RACE experiments were performed using primers derived of predicted exons especially in a 2.2 kb EcoRI fragment about 20 kb centromeric of the repeats. So far, only non-4q35 genes could be identified. Altogether, our results support other recent studies indicating that the FSHD gene is most likely not encoded by the 3.3 kb repeated units. Moreover, the region centromeric of these repeats appeared to contain abundant repetitive sequences and homologies to several other chromosomes, complicating the identification of the FSHD gene.

  16. Hairpin formation within the enhancer region of the human enkephalin gene

    SciTech Connect

    McMurray, C.T.; Douglass, J.O. ); Wilson, W.D. )

    1991-01-15

    The 3{prime},5{prime}-cyclic adenosine monophosphate (cAMP)-inducible enhancer of the human enkephaline gene is located within an imperfect palindrom of 23 base pairs. The authors have found that a 23-base-pair oligonucleotide duplex containing the enhancer undergoes a reversible conformational transition from the duplex to two individual hairpin structures each formed from one strand of the duplex. Each individual hairpin forms with mismatched base pairs, one containing two GT pairs and the other containing two AC pairs. The conformational transition is stabilized by proton transfer to the hairpin containing AC mismatched pairs. The unique physical and thermodynamic properties of the enkephalin enhancer DNA suggest a model in which DNA secondary structure within the enhancer region plays and active role incAMP-inducible activation of the human enkephalin gene via formation of cruciform structures.

  17. Nucleotide diversity analysis highlights functionally important genomic regions

    PubMed Central

    Tatarinova, Tatiana V.; Chekalin, Evgeny; Nikolsky, Yuri; Bruskin, Sergey; Chebotarov, Dmitry; McNally, Kenneth L.; Alexandrov, Nickolai

    2016-01-01

    We analyzed functionality and relative distribution of genetic variants across the complete Oryza sativa genome, using the 40 million single nucleotide polymorphisms (SNPs) dataset from the 3,000 Rice Genomes Project (http://snp-seek.irri.org), the largest and highest density SNP collection for any higher plant. We have shown that the DNA-binding transcription factors (TFs) are the most conserved group of genes, whereas kinases and membrane-localized transporters are the most variable ones. TFs may be conserved because they belong to some of the most connected regulatory hubs that modulate transcription of vast downstream gene networks, whereas signaling kinases and transporters need to adapt rapidly to changing environmental conditions. In general, the observed profound patterns of nucleotide variability reveal functionally important genomic regions. As expected, nucleotide diversity is much higher in intergenic regions than within gene bodies (regions spanning gene models), and protein-coding sequences are more conserved than untranslated gene regions. We have observed a sharp decline in nucleotide diversity that begins at about 250 nucleotides upstream of the transcription start and reaches minimal diversity exactly at the transcription start. We found the transcription termination sites to have remarkably symmetrical patterns of SNP density, implying presence of functional sites near transcription termination. Also, nucleotide diversity was significantly lower near 3′ UTRs, the area rich with regulatory regions. PMID:27774999

  18. Nucleotide diversity analysis highlights functionally important genomic regions.

    PubMed

    Tatarinova, Tatiana V; Chekalin, Evgeny; Nikolsky, Yuri; Bruskin, Sergey; Chebotarov, Dmitry; McNally, Kenneth L; Alexandrov, Nickolai

    2016-10-24

    We analyzed functionality and relative distribution of genetic variants across the complete Oryza sativa genome, using the 40 million single nucleotide polymorphisms (SNPs) dataset from the 3,000 Rice Genomes Project (http://snp-seek.irri.org), the largest and highest density SNP collection for any higher plant. We have shown that the DNA-binding transcription factors (TFs) are the most conserved group of genes, whereas kinases and membrane-localized transporters are the most variable ones. TFs may be conserved because they belong to some of the most connected regulatory hubs that modulate transcription of vast downstream gene networks, whereas signaling kinases and transporters need to adapt rapidly to changing environmental conditions. In general, the observed profound patterns of nucleotide variability reveal functionally important genomic regions. As expected, nucleotide diversity is much higher in intergenic regions than within gene bodies (regions spanning gene models), and protein-coding sequences are more conserved than untranslated gene regions. We have observed a sharp decline in nucleotide diversity that begins at about 250 nucleotides upstream of the transcription start and reaches minimal diversity exactly at the transcription start. We found the transcription termination sites to have remarkably symmetrical patterns of SNP density, implying presence of functional sites near transcription termination. Also, nucleotide diversity was significantly lower near 3' UTRs, the area rich with regulatory regions.

  19. cDNA sequence, genomic organization, and evolutionary conservation of a novel gene from the WAGR region

    SciTech Connect

    Schwartz, F.; Eisenman, R.; Knoll, J.; Bruns, G.

    1995-09-20

    A new gene (239FB) with predominant and differential expression in fetal brain has recently been isolated from a chromosome 11p13-p14 boundary area near FSHB. The corresponding mRNA has an open reading frame of 294 amino acids, a 3` untranslated region of 1247 nucleotides, and a highly GC-rich 5` untranslated region. The coding and 3` UT sequence is specified by 6 exons within nearly 87 kb of isolated genomic locus. The 5` end region of the transcript maps adjacent to the only genomically defined CpG island in a chromosomal subregion that may be associated with part of the mental retardation of some WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome patients. In addition to nucleotide and amino acid similarity to an EST from a normalized infant brain cDNA library, the predicted protein has extensive similarity to Caenorhbditis elegans polypeptides of, as yet, unknown function. The 239FB locus is, therefore, likely part of a family of genes with two members expressed in human brain. The extensive conservation of the predicted protein suggests a fundamental function of the gene product and will enable evaluation of the role of the 239FB gene in neurogenesis in model organisms. 48 refs., 4 figs., 1 tab.

  20. Altering the expression in mice of genes by modifying their 3' regions.

    PubMed

    Kakoki, Masao; Tsai, Yau-Sheng; Kim, Hyung-Suk; Hatada, Seigo; Ciavatta, Dominic J; Takahashi, Nobuyuki; Arnold, Larry W; Maeda, Nobuyo; Smithies, Oliver

    2004-04-01

    Polymorphic differences altering expression of genes without changing their products probably underlie human quantitative traits affecting risks of serious diseases, but methods for investigating such quantitative differences in animals are limited. Accordingly, we have developed a procedure for changing the expression in mice of chosen genes over a 100-fold range while retaining their chromosomal location and transcriptional controls. To develop the procedure, we first dissected the effects in embryonic stem (ES) cells of elements within and downstream of the 3' untranslated region (UTR) of a single copy transgene at the Hprt locus. As expected, protein expression varied with the steady-state level and half-life of the mRNA. The rank order of expression with various tested 3' regions is the same in ES cells, and in cardiomyocytes and trophoblastocytes derived from them. In mice having two functionally different native genes with modified 3'UTRs, the desired expression was obtained.

  1. Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO)

    SciTech Connect

    Shiang, R. ); Lidral, A.C.; Ardinger, H.H.; Murray, J.C.; Romitti, P.A.; Munger, R.G.; Buetow, K.H.

    1993-10-01

    Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. The authors carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3[prime] untranslated region of the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using X[sup 2] analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3[prime] untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P. 46 refs., 3 figs., 3 tabs.

  2. The effects on the expression of. beta. -lactamase by targeted insertion of a Kirsten murine leukemia virus variant into the coding region of the gene

    SciTech Connect

    Dias-Ferrao, V.P.T.

    1988-01-01

    The product of this plasmid gene protects bacteria from the antibiotic, ampicillin. When the Kirsten murine leukemia virus variant DNA (MuLV-K-Vd) was inserted into the Pst 1 site of the {beta}-lactamase gene, the transformed bacteria (E. coli, DH5) were resistant to ampicillin. The purpose of this study is to explain the presence of a functional {beta}-lactamase gene with additional nucleotides inserted into the coding region of the gene. The recombinant plasmid codes for a functional {beta}-lactamase. Northern blot analysis of RNA using a {sup 32}P-labelled 16{sup mer} oligonucleotide as a probe revealed the {beta}-lactamase transcript from the recombinant plasmid to be shorter than the transcript from the wild-type {beta}-lactamase gene. Also, greater levels of {beta}-lactamase mRNA were present in cells containing the recombinant plasmid compared to those containing the wild-type plasmid. Restriction enzyme mapping indicated that the 3{prime} end of MuLV-K-Vd insert contains sequences of {beta}-lactamase. Nucleic acid sequencing substantiated the hybridization data that {beta}-lactamase sequences are present in the 3{prime} end of MuLV-K-Vd. However, exact sequence homology is not evident.

  3. High sequence turnover in the regulatory regions of the developmental gene hunchback in insects.

    PubMed

    Hancock, J M; Shaw, P J; Bonneton, F; Dover, G A

    1999-02-01

    Extensive sequence analysis of the developmental gene hunchback and its 5' and 3' regulatory regions in Drosophila melanogaster, Drosophila virilis, Musca domestica, and Tribolium castaneum, using a variety of computer algorithms, reveals regions of high sequence simplicity probably generated by slippage-like mechanisms of turnover. No regions are entirely refractory to the action of slippage, although the density and composition of simple sequence motifs varies from region to region. Interestingly, the 5' and 3' flanking regions share short repetitive motifs despite their separation by the gene itself, and the motifs are different in composition from those in the exons and introns. Furthermore, there are high levels of conservation of motifs in equivalent orthologous regions. Detailed sequence analysis of the P2 promoter and DNA footprinting assays reveal that the number, orientation, sequence, spacing, and protein-binding affinities of the BICOID-binding sites varies between species and that the 'P2' promoter, the nanos response element in the 3' untranslated region, and several conserved boxes of sequence in the gene (e.g., the two zinc-finger regions) are surrounded by cryptically-simple-sequence DNA. We argue that high sequence turnover and genetic redundancy permit both the general maintenance of promoter functions through the establishment of coevolutionary (compensatory) changes in cis- and trans-acting genetic elements and, at the same time, the possibility of subtle changes in the regulation of hunchback in the different species.

  4. Multiple pathways for steel regulation suggested by genomic and sequence analysis of the murine Steel gene

    SciTech Connect

    Bedell, M.A.; Copeland, N.G.; Jenkins, N.A.

    1996-03-01

    The Steel (Sl) locus encodes mast cell growth factor (Mgf) that is required for the development of germ cells, hematopoietic cells and melanocytes. Although the expression patterns of the Mgf gene are well characterized, little is known of the factors which regulate its expression. Here, we describe the cloning and sequence of the full-length transcription unit and the 5{prime} flanking region of the murine Mgf gene. The full-length Mgf mRNA consists of a short 5{prime} untranslated region (UTR), a 0.8-kb ORF and a long 3{prime} UTR. A single transcription initiation site is used in a number of mouse tissues and is located just downstream of binding sites for several known transcription factors. In the 5{prime} UTR, two ATGs were found upstream of the initiator methionine and are conserved among different species, suggesting that Mgf may be translationally regulated. At least two Mgf mRNAs are produced by alternative use of polyadenylation sites, but numerous other potential polyadenylation sites were found in the 3{prime} UTR. In addition, the 3{prime} UTR contains numerous sequence motifs that may regulate Mgf mRNA stability. These studies suggest multiple ways in which expression of Mgf may be regulated. 39 refs., 4 figs.

  5. Genomic organization of the neurofibromatosis 1 gene (NF1)

    SciTech Connect

    Li, Y.; O`Connell, P.; Huntsman Breidenbach, H.

    1995-01-01

    Neurofibromatosis 1 maps to chromosome band 17q11.2, and the NF1 locus has been partially characterized. Even though the full-length NF1 cDNA has been sequenced, the complete genomic structure of the NF1 gene has not been elucidated. The 5{prime} end of NF1 is embedded in a CpG island containing a NotI restriction site, and the remainder of the gene lies in the adjacent 350-kb NotI fragment. In our efforts to develop a comprehensive screen for NF1 mutations, we have isolated genomic DNA clones that together harbor the entire NF1 cDNA sequence. We have identified all intron-exon boundaries of the coding region and established that it is composed of 59 exons. Furthermore, we have defined the 3{prime}-untranslated region (3{prime}-UTR) of the NF1 gene; it spans approximately 3.5 kb of genomic DNA sequence and is continuous with the stop codon. Oligonucleotide primer pairs synthesized from exon-flanking DNA sequences were used in the polymerase chain reaction with cloned, chromosome 17-specific genomic DNA as template to amplify NF1 exons 1 through 27b and the exon containing the 3{prime}-UTR separately. This information should be useful for implementing a comprehensive NF1 mutation screen using genomic DNA as template. 41 refs., 3 figs., 2 tabs.

  6. Identification of candidates for human disease genes using large-scale PCR mapping of gene-based STSs

    SciTech Connect

    Berry, R.; Stevens, T.J.; Wilcox, A.S.

    1994-09-01

    We have developed a strategy for the rapid identification of possible human disease/syndrome genes. Using this procedure we found candidates for 45 human disease/syndrome genes from the first 200 genes mapped. New human genes are identified through automated single-pass sequencing into the 3{prime} untranslated (3{prime}UT) regions of human cDNAs. Primers derived from the 3{prime}UT region sequences, representing gene-based STSs, are used for PCR analyses of the CEPH megabase YAC DNA pools. With this approach {approximately}18,000 megabase YACs can be screened and a single YAC identified using only 52 PCR reactions. The YAC localization in conjunction with other mapping approaches, such as PCR mapping to chromosomes by means of somatic hybrids, allows mapping to chromosomal band locations. In this manner, each gene can be associated with its own STS which in turn specifies both a corresponding genomic clone and a specific location in the genome. These locations can be compared to purported locations of disease genes listed in Online Mendelian Inheritance in Man. Using our current collection of >3,000 human brain cDNA sequences as a resource, we have carried out a proof of principle study in which {approximately}200 cDNAs were mapped to YACs within a few months. Appropriate scale up of this strategy could permit mapping of most human genes and identification of many candidate disease genes over the next few years.

  7. Transgenic mouse model of hemifacial microsomia: Cloning and characterization of insertional mutation region on chromosome 10

    SciTech Connect

    Naora, Hiroyuki; Otani, Hiroki; Tanaka, Osamu

    1994-10-01

    The 643 transgenic mouse line carries an autosomal dominant insertional mutation that results in hemifacial microsomia (HFM), including microtia and/or abnormal biting. In this paper, we characterize the transgene integration site in transgenic mice and preintegration site of wildtype mice. The locus, designated Hfm (hemifacial microsomia-associated locus), was mapped to chromosome 10, B1-3, by chromosome in situ hybridization. We cloned the transgene insertion site from the transgenic DNA library. By using the 5{prime} and 3{prime} flanking sequences, the preintegration region was isolated. The analysis of these regions showed that a deletion of at least 23 kb DNA occurred in association with the transgene integration. Evolutionarily conserved regions were detected within and beside the deleted region. The result of mating between hemizygotes suggests that the phenotype of the homozygote is lethality in the prenatal period. These results suggests that the Hfm locus is necessary for prenatal development and that this strain is a useful animal model for investigating the genetic predisposition to HFM in humans.

  8. The transcriptionally active regions in the genome of Bacillus subtilis

    PubMed Central

    Rasmussen, Simon; Nielsen, Henrik Bjørn; Jarmer, Hanne

    2009-01-01

    The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome-wide expression during mid-exponential growth on rich (LB) and minimal (M9) medium. The identified TARs account for 77.3% of the genes as they are currently annotated and additionally we find 84 putative non-coding RNAs (ncRNAs) and 127 antisense transcripts. One ncRNA, ncr22, is predicted to act as a translational control on cstA and an antisense transcript was observed opposite the housekeeping sigma factor sigA. Through this work we have discovered a long conserved 3′ untranslated region (UTR) in a group of membrane-associated genes that is predicted to fold into a large and highly stable secondary structure. One of the genes having this tail is efeN, which encodes a target of the twin-arginine translocase (Tat) protein translocation system. PMID:19682248

  9. Chaos Region

    NASA Image and Video Library

    2015-09-10

    In the center of this 300-mile (470-kilometer) wide image of Pluto from NASA's New Horizons spacecraft is a large region of jumbled, broken terrain on the northwestern edge of the vast, icy plain informally called Sputnik Planum, to the right. The smallest visible features are 0.5 miles (0.8 kilometers) in size. This image was taken as New Horizons flew past Pluto on July 14, 2015, from a distance of 50,000 miles (80,000 kilometers). http://photojournal.jpl.nasa.gov/catalog/PIA19934

  10. NcoI RFLP at the creatine kinase-muscle type gene locus (CKMM, chromosome 19)

    SciTech Connect

    Coerwinkel-Driessen, M.; Schepens, J.; van Zandvoort, P.; van Oost, B.; Mariman, E.; Wieringa, B. )

    1988-09-12

    A 3.2 kbp human genomic DNA fragment (BamHI-Sau3A) of the 3{prime} untranslated and 3{prime} flanking region of the CKMM gene was isolated and subcloned into the BamHI site of vector pSP64. The CKMM 3{prime}-probe identifies a 2-allele polymorphism with bands at 2.3 and 1.0 kbp (allele A) and 3.3 kbp (allele B). In addition a weak constant 4.2 kbp band is observed. This probe also detects a 2-allele TaqI RFLP reported previously, as either a 4.3 kbp (A) or a 4.2 kbp (B) band. The CKMM locus previously has been assigned to 19q13.2-q13.3. By Southern blot analysis of human-rodent somatic cell hybrids containing unique subregional fragments of chromosome 19 of man the authors have assigned the gene to 19q13.2. Co-dominant segregation was observed in 8 families with 3 generations.

  11. A novel haplotype in ApoAI-CIII-AIV gene region is detrimental to Northwest Indians with coronary heart disease.

    PubMed

    Singh, Puneetpal; Singh, Monica; Kaur, T P; Grewal, S S

    2008-11-28

    The present study investigated the genetic variation of 3' flanking region of ApoA-I (PstI), 3' untranslated region of ApoC-III (SstI) and intron 2 of ApoA-IV (XbaI) in 193 angiographically diagnosed CHD patients and 150 CHD negative controls of Punjab, Northwest India. Haplotype analysis reveals that P2-S2-X1 is a susceptibility haplotype that confers the risk of CHD (OR 2.33, CI 1.08-4.38, P<0.05), which exacerbates (OR 2.61, CI 1.23-5.92, P<0.01) after adjustment with the confounders. This exacerbating effect of P2-S2-X1 may umpire significant higher levels of TG, LDL/HDL ratio and lower levels of HDL in CHD patients.

  12. Identification and genetic mapping of a homeobox gene to the 4p16.1 region of human chromosome 4.

    PubMed

    Stadler, H S; Padanilam, B J; Buetow, K; Murray, J C; Solursh, M

    1992-12-01

    A human craniofacial cDNA library was screened with a degenerate oligonucleotide probe based on the conserved third helix of homeobox genes. From this screening, we identified a homeobox gene, H6, which shared only 57-65% amino acid identity to previously reported homeodomains. H6 was physically mapped to the 4p16.1 region by using somatic cell hybrids containing specific deletions of human chromosome 4. Linkage data from a single-stranded conformational polymorphism derived from the 3' untranslated region of the H6 cDNA placed this homeobox gene more than 20 centimorgans proximal of the previously mapped HOX7 gene on chromosome 4. Identity comparisons of the H6 homeodomain with previously reported homeodomains reveal the highest identities to be with the Nk class of homeobox genes in Drosophila melanogaster.

  13. Identification and genetic mapping of a homeobox gene to the 4p16.1 region of human chromosome 4.

    PubMed Central

    Stadler, H S; Padanilam, B J; Buetow, K; Murray, J C; Solursh, M

    1992-01-01

    A human craniofacial cDNA library was screened with a degenerate oligonucleotide probe based on the conserved third helix of homeobox genes. From this screening, we identified a homeobox gene, H6, which shared only 57-65% amino acid identity to previously reported homeodomains. H6 was physically mapped to the 4p16.1 region by using somatic cell hybrids containing specific deletions of human chromosome 4. Linkage data from a single-stranded conformational polymorphism derived from the 3' untranslated region of the H6 cDNA placed this homeobox gene more than 20 centimorgans proximal of the previously mapped HOX7 gene on chromosome 4. Identity comparisons of the H6 homeodomain with previously reported homeodomains reveal the highest identities to be with the Nk class of homeobox genes in Drosophila melanogaster. Images PMID:1360670

  14. Photodissociation Regions

    NASA Technical Reports Server (NTRS)

    Hollenbach, David J.; DeVincenzi, Donald L. (Technical Monitor)

    2000-01-01

    The interstellar medium of galaxies is the reservoir out of which stars are born and into which stars inject newly created elements as they age. The physical properties of the interstellar medium are governed in part by the radiation emitted by these stars. Far-ultraviolet (6 eV< hNu < 13.6 eV) photons from massive stars dominate the heating and influence the chemistry of the neutral atomic gas and much of the molecular gas in galaxies. Predominantly neutral regions of the interstellar medium in which the heating and chemistry are regulated by far ultraviolet photons are termed Photodissociation Regions (PDRs). These regions are the origin of most of the non-stellar infrared (IR) and the millimeter and submillimeter CO emission from galaxies. The importance of PDRs has become increasingly apparent with the advances in IR and submillimeter astronomy. The IR emission from PDRs includes fine structure lines of C, C(+) and O; rovibrational lines of H2; rotational lines of CO; broad mid-IR features of polycyclic aromatic hydrocarbons; and a luminous underlying IR continuum from interstellar dust. The transition of H to H2 and C(+) to CO occurs within PDRs. Comparison of observations with theoretical models of PDRs enables one to determine the density and temperature structure, the elemental abundances, the level of ionization, and the radiation field. PDR models have been applied to interstellar clouds near massive stars, planetary nebulae, red giant outflows, photoevaporating planetary disks around newly formed stars, diffuse clouds, the neutral intercloud medium, and molecular clouds in the interstellar radiation field-in summary, much of the interstellar medium in galaxies. Theoretical PDR models explain the observed correlations of the [CII] 158, micrometers with the CO J=1-0 emission, the CO J=1-0 luminosity with the interstellar molecular mass, and the [CII] 158 micrometers plus [OI] 63 micrometers luminosity with the IR continuum luminosity. On a more global

  15. Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates.

    PubMed

    Chang, Perng-Kuang; Horn, Bruce W; Dorner, Joe W

    2005-11-01

    Aspergillus flavus populations are genetically diverse. Isolates that produce either, neither, or both aflatoxins and cyclopiazonic acid (CPA) are present in the field. We investigated defects in the aflatoxin gene cluster in 38 nonaflatoxigenic A. flavus isolates collected from southern United States. PCR assays using aflatoxin-gene-specific primers grouped these isolates into eight (A-H) deletion patterns. Patterns C, E, G, and H, which contain 40 kb deletions, were examined for their sequence breakpoints. Pattern C has one breakpoint in the cypA 3' untranslated region (UTR) and another in the verA coding region. Pattern E has a breakpoint in the amdA coding region and another in the ver1 5'UTR. Pattern G contains a deletion identical to the one found in pattern C and has another deletion that extends from the cypA coding region to one end of the chromosome as suggested by the presence of telomeric sequence repeats, CCCTAATGTTGA. Pattern H has a deletion of the entire aflatoxin gene cluster from the hexA coding region in the sugar utilization gene cluster to the telomeric region. Thus, deletions in the aflatoxin gene cluster among A. flavus isolates are not rare, and the patterns appear to be diverse. Genetic drift may be a driving force that is responsible for the loss of the entire aflatoxin gene cluster in nonaflatoxigenic A. flavus isolates when aflatoxins have lost their adaptive value in nature.

  16. Solution confirmation of the (-)-trans-anti-5-Methylchrysene-dG adduct oppposite dC in a DNA duplex: DNA bending associated with wedging of the Methyl group of 5-Methylchrysene to the 3{prime}-side of the modification site

    SciTech Connect

    Cosman, M.; Patel, D.J.

    1995-05-09

    This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(Cl-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11){sm_bullet}d(G12-G13-T14-A15-G16-C17-G 18-A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG{sm_bullet}dC 11-mer duplex]. This adduct is derived from the trans addition at C{sup 4} of (-)-anti-1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4-tetrahydro-5-methylchrysene [(-)-anti-5-MeCDE] to the N{sup 2} position of dG6 in this duplex sequence. The 5-methyl group is located adjacent to the MC(C{sup 4}) binding site, with these groups juxtaposed in a sterically crowded bay region in the adduct duplex. The 5-methylchrysenyl and the nucleic acid exchangeable and nonexchangeable protons were assigned following analysis of two-dimensional NMR data sets in H{sub 2}O and D{sub 2}O buffer solution. The solution structure of the trans-anti-[MC]dG{sm_bullet}dC 11-mer duplex has been determined by incorporating DNA-DNA and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the [MC]dG6{sm_bullet}dC17 base pair and flanking dC5{sm_bullet}dG18 and dC7{sm_bullet}dG16 base pairs retain Watson-Crick alignments upon adduct formation. 61 refs., 9 figs., 2 tabs.

  17. MICB gene diversity and balancing selection on its promoter region in Yao population in southern China.

    PubMed

    Chen, Xiang; Liu, Xuexiang; Wei, Xiaomou; Meng, Yuming; Liu, Limin; Qin, Shini; Liu, Yanyu; Dai, Shengming

    2016-12-01

    To comprehensively examine the MICB gene polymorphism and identify its differences in Chinese Yao population from other ethnic groups, we investigated the polymorphism in the 5'-upstream regulation region (5'-URR), coding region (exons 2-4), and the 3'-untranslated region (3'-UTR) of MICB gene by using PCR-SBT method in 125 healthy unrelated Yao individuals in Guangxi Zhuang Autonomous Region. Higher polymorphism was observed in the 5'-URR, nine single nucleotide polymorphisms (SNPs) and a two base pairs deletion at position -139/-138 were found in our study. Only five different variation sites, however, were detected in exons 2-4 and three were observed in the 3'-UTR. The minor allele frequencies of all variants were greater than 5%, except for rs3828916, rs3131639, rs45627734, rs113620316, rs779737471, and the variation at position +11803 in the 3'-UTR. The first nine SNPs of 5'-URR and rs1065075, rs1051788 of the coding region showed significant linkage disequilibrium with each other. Ten different MICB extended haplotypes (EH) encompassing the 5'-URR, exons 2-4, and 3'-UTR were found in this population, and the most frequent was EH1 (23.2%). We provided several evidences for balancing selection effect on the 5'-URR of MICB gene in Yao population. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  18. Identification, characterization and functional analysis of regulatory region of nanos gene from half-smooth tongue sole (Cynoglossus semilaevis).

    PubMed

    Huang, Jinqiang; Li, Yongjuan; Shao, Changwei; Wang, Na; Chen, Songlin

    2017-03-25

    The nanos gene encodes an RNA-binding zinc finger protein, which is required in the development and maintenance of germ cells. However, there is very limited information about nanos in flatfish, which impedes its application in fish breeding. In this study, we report the molecular cloning, characterization and functional analysis of the 3'-untranslated region of the nanos gene (Csnanos) from half-smooth tongue sole (Cynoglossus semilaevis), which is an economically important flatfish in China. The 1233-bp cDNA sequence, 1709-bp genomic sequence and flanking sequences (2.8-kb 5'- and 1.6-kb 3'-flanking regions) of Csnanos were cloned and characterized. Sequence analysis revealed that CsNanos shares low homology with Nanos in other species, but the zinc finger domain of CsNanos is highly similar. Phylogenetic analysis indicated that CsNanos belongs to the Nanos2 subfamily. Csnanos expression was widely detected in various tissues, but the expression level was higher in testis and ovary. During early development and sex differentiation, Csnanos expression exhibited a clear sexually dimorphic pattern, suggesting its different roles in the migration and differentiation of primordial germ cells (PGCs). Higher expression levels of Csnanos mRNA in normal females and males than in neomales indicated that the nanos gene may play key roles in maintaining the differentiation of gonad. Moreover, medaka PGCs were successfully labeled by the microinjection of synthesized mRNA consisting of green fluorescence protein and the 3'-untranslated region of Csnanos. These findings provide new insights into nanos gene expression and function, and lay the foundation for further study of PGC development and applications in tongue sole breeding.

  19. Retroviral and psuedogene insertion sites reveal the lineage of human salivary and pancreatic amylase genes from a single gene during primate evolution

    SciTech Connect

    Samuelson, L.C.; Snow, C.M.; Meisler, M.H. . Dept. of Human Genetics); Wiebauer, K. )

    1990-06-01

    The authors have analyzed the junction regions of inserted elements within the human amylase gene complex. This complex contains five genes which are expressed at high levels either in the pancreas or in the parotid gland. The proximal 5{prime}-flanking regions of these genes contain two inserted elements. A {gamma}-actin pseudogene is located at a position 20 base pairs upstream of the first coding exon. All of the amylase genes contain this insert. The subsequent insertion of an endogenous retrovirus interrupted the {gamma}-actin pseudogene within its 3{prime}-untranslated region. Nucleotide sequence analysis of the inserted elements associated with each of the five human amylase genes has revealed a series of molecular events during the recent history of this gene family. The data indicate that the entire gene family was generated during primate evolution from one ancestral gene copy and that the retroviral insertion activated a cryptic promoter.

  20. Different mRNAs code for dopa decarboxylase in tissues of neuronal and nonneuronal origin

    SciTech Connect

    Krieger, M.; Coge, F.; Gros, F.; Thibault, J. )

    1991-03-15

    A cDNA clone for dopa decarboxylase has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5{prime} end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Droxophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5{prime} untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3{prime} untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5{prime} untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.

  1. Genetic and physical mapping of the Treacher Collins syndrome locus with respect to loci in the chromosome 5q3 region

    SciTech Connect

    Jabs, E.W.; Li, Xiang; Coss, C.; Taylor, E. ); Lovett, M. ); Yamaoka, L.H.; Speer, M.C. ); Cadle, R.; Hall, B. ); Brown, K. )

    1993-10-01

    Treacher Collins syndrome is an autosomal dominant, craniofacial developmental disorder, and its locus (TCOF1) has been mapped to chromosome 5q3. To refine the location of the gene within this region, linkage analysis was performed among the TCOF1 locus and 12 loci (IL9, FGFA, GRL, D5S207, D5S210, D5S376, CSF1R, SPARC, D5S119, D5S209, D5S527, FGFR4) in 13 Treacher Collins syndrome families. The highest maximum lod score was obtained between loci TCOF1 and D5S210 (Z = 10.52; [theta] = 0.02 [+-] 0.07). The best order, IL9-GRL-D5S207/D5S210-CSF1R-SPARC-D5S119, and genetic distances among these loci were determined in the 40 CEPH families by multipoint linkage analysis. YAC clones were used to establish the order of loci, centromere-5[prime]GRL3[prime]-D5S207-D5S210-D5S376-CSF1R-SPARC-D5S119-telomere. By combining known physical mapping data with ours, the order of chromosome 5q3 markers is centomere-IL9-FGFA-5[prime]GRL3[prime]-D5s207-D5S210-D5S376-CSF1R-SPARC-D5S119-D5S209-FGFR4-telomere. Based on this order, haplotype analysis suggests that the TCOF1 locus resides distal CSF1R and proximal to SPARC within a region less than 1 Mb in size. 29 refs., 2 figs., 2 tabs.

  2. A 3 Mb YAC contig in the region of Usher Ib on chromosome 11q

    SciTech Connect

    Kelley, P.M.; Overbeck, L.; Weston, M.

    1994-09-01

    Under syndrome type Ib, a recessive disorder characterized by deafness, retinitis pigmentosa, and vestibular dysfunction has been mapped to chromosome 11q13. A 3 Mb YAC contig has been constructed covering the critical region of Usher Ib and spanning over eight loci: D11S1321, D11S527, D11S533, OMP, D11S906, D11S911, D11S937, and D11S918. This contig was constructed by PCR screening using the above described DNA markers of the CEPH mega YAC library. Additional YACs were identified by data presented in the Genethon physical map. A long-range restriction map has been constructed from both YAC and genomic DNA using STS markers as probes. Cosmid libraries from a subset of YACs have been screened for the location of CpG islands. In addition, potential transcribed regions have been identified by 3{prime} exon trapping of cosmid pools and placed on the YAC physical map.

  3. Absence of mutations in the interspecies conserved regions of the CFTR promoter region in cystic fibrosis (CF) and CF related patients.

    PubMed Central

    Verlingue, C; Vuillaumier, S; Mercier, B; Le Gac, M; Elion, J; Férec, C; Denamur, E

    1998-01-01

    This study was aimed at testing if a 5.2 kb untranslated region on both sides of the first CFTR exon, shown to contain regulatory elements, could carry mutations responsible for cystic fibrosis (CF) or CF related phenotypes. Selection of the DNA segments studied within this region was based upon the identification of conserved sequences throughout evolution (phylogenetic footprints, PFs). Comparison of the CFTR sequences in eight species representing four orders of mammals (man, gibbon, rhesus monkey, squirrel, monkey, rabbit, cow, rat, and mouse) identified four clusters of PFs within the 3.9 kb of DNA sequence upstream from the initiation codon, as well as two nearby PFs at +1 kb within intron 1. Six DNA segments containing PFs were scanned for mutations by denaturing gradient gel electrophoresis (DGGE) in patients with CF (n = 29), congenital bilateral absence of the vas deferens (n = 143), or disseminated bronchiectasis (n = 33), for whom only one or no mutations had been identified despite extensive DGGE analysis of the 27 CFTR exons and exon/intron boundaries. Only one polymorphism (-966 T-->G) was identified with a frequency of 2.2% and no other sequence variations were found. This study reinforces the idea that the promoter region in the CFTR is not frequently mutated. PMID:9507393

  4. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    SciTech Connect

    Fukumitsu, Hidefumi; Soumiya, Hitomi; Furukawa, Shoei

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CFIm25 knockdown promoted NGF-induced neurite out growth from PC12 cells. Black-Right-Pointing-Pointer Depletion of CFIm25 did not influence the morphology of proliferating PC12 cells. Black-Right-Pointing-Pointer CFIm regulated NGF-induced neurite outgrowth via coordinating RhoA activity. Black-Right-Pointing-Pointer CFIm25 knockdown increase the number of primary dendrites of hippocampal neurons. -- Abstract: Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3 Prime -untranslated region (3 Prime -UTR), producing mRNAs with variable 3 Prime ends. Because 3 Prime -UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25 kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

  5. Rarity of DNA sequence alterations in the promoter region of the human androgen receptor gene.

    PubMed

    Cabral, D F; Santos, A; Ribeiro, M L; Mesquita, J C; Carvalho-Salles, A B; Hackel, C

    2004-12-01

    The human androgen receptor (AR) gene promoter lies in a GC-rich region containing two principal sites of transcription initiation and a putative Sp1 protein-binding site, without typical "TATA" and "CAAT" boxes. It has been suggested that mutations within the 5'untranslated region (5'UTR) may contribute to the development of prostate cancer by changing the rates of gene transcription and/or translation. In order to investigate this question, the aim of the present study was to search for the presence of mutations or polymorphisms at the AR-5'UTR in 92 prostate cancer patients, where histological diagnosis of adenocarcinoma was established in specimens obtained from transurethral resection or after prostatectomy. The AR-5'UTR was amplified by PCR from genomic DNA samples of the patients and of 100 healthy male blood donors, included as controls. Conformation-sensitive gel electrophoresis was used for DNA sequence alteration screening. Only one band shift was detected in one individual from the blood donor group. Sequencing revealed a new single nucleotide deletion (T) in the most conserved portion of the promoter region at position +36 downstream from the transcription initiation site I. Although the effect of this specific mutation remains unknown, its rarity reveals the high degree of sequence conservation of the human androgen promoter region. Moreover, the absence of detectable variation within the critical 5'UTR in prostate cancer patients indicates a low probability of its involvement in prostate cancer etiology.

  6. Joint Electron Beam Commonality Experiments on Blackjack 3 and 3 Prime. Part I. Machine Characterizations

    DTIC Science & Technology

    1979-12-31

    0.32 Ta/PMMA 0.09 X-cut Quartz 151 Fused Silica 130 PMMA 32 aunits of kbar/(mm/us). blnterface, stress passing from metal to backer. cTransmitted stress...218 774 ., -41’ -+&~-OE-T. ttefP-COS-S-BEP-T -- NCRP -COS-E- i(Gm/c .i --.I7= - . - L �.. 4-, 5 52"-02 r " .,D-0 .. .. ,. 8 --. "񓃮

  7. Skylab 3 prime crew participate in water egress simulations at JSC

    NASA Image and Video Library

    1973-05-01

    S73-27787 (1 May 1973) --- The three members of the prime crew of the second manned Skylab mission participate in prelaunch training, specifically water egress simulations, at the Johnson Space Center (JSC), Houston. They are, left to right, astronaut Alan J. Bean, commander; scientist-astronaut Owen K. Garriott, science pilot; and astronaut Jack R. Lousma, pilot. This training took place in JSC?s Building 220 on May 1, 1973. Photo credit: NASA

  8. Stereoselective formation of a 2 prime (3 prime)- aminoacyl ester of a nucleotide

    NASA Technical Reports Server (NTRS)

    Weber, A. L.

    1986-01-01

    Reaction of DL-series and adenosine-5-phosphorimidazolide in the presence of adenosine-5'-(0-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester, 2'(3')-0-seryl-adenosine-5'-(0-methylphosphate). The enantiomeric excess of D-serine incorporated into 2'(3')-0-seryl-adenosine-5'-(0-methylphosphate) was about 9%. Adenylyl-(5->N)-serine and an unknown product also incorporated an excess of D-serine, however, seryl-serine showed an excess of L-serine. The relationship of these results to the origin of the biological pairing of L-amino acids and nucleotides containing D-ribose is discussed.

  9. 2[prime] and 3[prime] Carboranyl uridines and their diethyl ether adducts

    DOEpatents

    Soloway, A.H.; Barth, R.F.; Anisuzzaman, A.K.; Alam, F.; Tjarks, W.

    1992-12-15

    A process is described for preparing carboranyl uridine nucleoside compounds and their diethyl ether adducts, which exhibit a tenfold increase in boron content over prior art boron containing nucleoside compounds. The carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of the compounds in methods for boron neutron capture therapy in mammalian tumor cells. No Drawings

  10. The 3 prime paradigm of the miR-200 family and other microRNAs

    PubMed Central

    Moschos, Sterghios; Spivack, Simon D

    2011-01-01

    The number of predicted human microRNAs in Sanger miRBase currently stands at over 1,000, with each of these in turn predicted to target numerous mRNAs. However, those microRNAs for which mRNA targets have been evaluated, verified and reported in the literature are still in the minority and the bulk of microRNA/mRNA interactions are yet to be confirmed. Confirmation of microRNA interaction with predicted mRNA targets represents a considerable undertaking, made more complex by potential synergistic effects of multiple microRNAs and the three possible outcomes (translational repression, degradation or a mixture of both). In addition, contrasting results obtained when either stably expressing or transiently transfecting members of the miR-200 family illustrate limitations in the verification methods currently in use. In this article we suggest that instead of allowing computational predictions to drive investigation, it would be desirable, when possible, to systematically evaluate microRNA targets using inducible, stable, ectopic expression. The advantage of stable lines ectopically expressing microRNA(s) is that they allow an analysis of changes to both the proteome and the transcriptome. This would allow verification of targets, improve the design of prediction algorithms and greatly increase our understanding of the outcome of microRNA/mRNA interaction. PMID:21242719

  11. The 3 prime paradigm of the miR-200 family and other microRNAs.

    PubMed

    Brock, Graham J; Moschos, Sterghios; Spivack, Simon D; Hurteau, Gregory J

    2011-03-01

    The number of predicted human microRNAs in Sanger miRBase currently stands at over a thousand, with each of these in turn predicted to target numerous mRNAs. However, those microRNAs for which mRNA targets have been evaluated, verified and reported in the literature are still in the minority and the bulk of microRNA/mRNA interactions are yet to be confirmed. Confirmation of microRNA interaction with predicted mRNA targets represents a considerable undertaking, made more complex by potential synergistic effects of multiple microRNAs and the three possible outcomes (translational repression, degradation or a mixture of both). In addition, contrasting results obtained when either stably expressing or transiently transfecting members of the miR-200 family illustrate limitations in the verification methods currently in use. In this article we suggest that instead of allowing computational predictions to drive investigation, it would be desirable, when possible, to systematically evaluate microRNA targets using inducible, stable, ectopic expression. The advantage of stable lines ectopically expressing microRNA(s) is that they allow an analysis of changes to both the proteome and the transcriptome. This would allow verification of targets, improve the design of prediction algorithms and greatly increase our understanding of the outcome of microRNA/mRNA interaction.

  12. Returning "Region" to World Regional Geography

    ERIC Educational Resources Information Center

    Rees, Peter W.; Legates, Margaret

    2013-01-01

    World regional geography textbooks rarely focus on the process of region formation, despite frequent calls to reincorporate a regional approach to teaching global geography. An instructional strategy using problem-based learning in a small honors section of a large world regional geography course is described. Using a hypothetical scenario…

  13. The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis.

    PubMed

    Clyde, Karen; Barrera, Julio; Harris, Eva

    2008-09-30

    Dengue virus (DENV) and West Nile virus (WNV) are members of the Flavivirus genus of positive-strand RNA viruses. RNA sequences and structures, primarily in the untranslated regions, have been shown to modulate flaviviral gene expression and genome replication. Previously, we demonstrated that a structure in the DENV coding region (cHP) enhances translation start codon selection and is required for viral replication. Here we further characterize the role of the cHP in the DENV life cycle. We demonstrate that the cHP is required for efficient viral RNA synthesis in a sequence-independent manner. Viruses with a disrupted cHP are rescued by a spontaneous compensatory mutation that restabilizes the structure. Furthermore, the cHP, which is predicted to be conserved among arthropod-borne flaviviruses, is required for WNV replication. We propose that the cHP is a multifunctional determinant of flavivirus replication, functioning in both translation and RNA synthesis.

  14. Ionospheric research. [E region, F region, D region

    NASA Technical Reports Server (NTRS)

    1975-01-01

    Progress is reported in the following areas: D-region theory; E and F-region; wave propagation; mass spectrometer measurements; and atmospheric reactions. Various supporting operations are included: design and construction of instrumentation; and programming.

  15. Identification of a missense mutation and several polymorphisms in the proenkephalin A gene of schizophrenic patients

    SciTech Connect

    Mikesell, M.J.; Sommer, S.S.; McMurray, C.T.

    1996-09-20

    Schizophrenia is a complex and severe disorder of unknown cause and pathophysiology. In this study, we examined the opioid hypothesis for schizophrenia at the molecular level, focusing on the dopamine-regulated proenkephalin A gene (chromosome 8q11.23-q12). We have screened 150 schizophrenic patients for sequence variations within the promoter region, entire coding sequence, and 3{prime}-untranslated region. We find one sequence change in a conserved amino acid that may be of functional significance. This mutation was found in a single schizophrenia patient but not in controls. Although several new, race-specific polymorphisms were identified, all other sequence changes appeared to be common polymorphisms, unlikely to contribute to the etiology of schizophrenia. 38 refs., 3 figs., 2 tabs.

  16. Mapping of the taurine transporter gene to mouse chromosome 6 and to the short arm of human chromosome 3

    SciTech Connect

    Patel, A.; Uhl, G.R.; Gregor, P.

    1995-01-01

    Transport proteins have essential functions in the uptake of neurotransmitters and neuromodulators. We have mapped the gene encoding the taurine transporter, Taut, to the central region of mouse chromosome 6. Analysis of a cross segregating the neurological mutant mnd2 excluded Taut as a candidate gene for this closely linked mutation. To map the human taurine transporter gene, TAUT, a sequence-tagged site (STS) corresponding to the 3{prime} untranslated region of the human cDNA was developed. TAUT was assigned to human chromosome 3 by typing this STS on a panel of somatic cell hybrids. Further analysis of a hybrid panel containing defined deletions of chromosome 3 suggested that TAUT maps to 3p21-p25. These data extend a conserved linkage group on mouse chromosome 6 and human chromosome 3p. Deletion of TAUT might contribute to some phenotypic features of the 3p{sup -} syndrome. 32 refs., 3 figs.

  17. The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome.

    PubMed

    Fu, H; Park, W; Yan, X; Zheng, Z; Shen, B; Dooner, H K

    2001-07-17

    The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3' untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

  18. Sequence analysis of the ERCC2 gene regions in human, mouse, and hamster reveals three linked genes

    SciTech Connect

    Lamerdin, J.E.; Stilwagen, S.A.; Ramirez, M.H.

    1996-06-15

    The ERCC2 (excision repair cross-complementing rodent repair group 2) gene product is involved in transcription-coupled repair as an integral member of the basal transcription factor BTF2/TFIIH complex. Defects in this gene can result in three distinct human disorders, namely the cancer-prone syndrome xeroderma pigmentosum complementation group D, trichothiodystrophy, and Cockayne syndrome. We report the comparative analysis of 91.6 kb of new sequence including 54.3 kb encompassing the human ERCC2 locus, the syntenic region in the mouse (32.6 kb), and a further 4.7 kb of sequence 3{prime} of the previously reported ERCC2 region in the hamster. In addition to ERCC2, our analysis revealed the presence of two previously undescribed genes in all three species. The first is centromeric (in the human) to ERCC2 and is most similar to the kinesin light chain gene in sea urchin. The second gene is telomeric (in the human) to ERCC2 and contains a motif found in ankyrins, some cell proteins, and transcription factors. Multiple EST matches to this putative new gene indicate that it is expressed in several human tissues, including breast. The identification and description of two new genes provides potential candidate genes for disorders mapping to this region of 19q13.2. 42 refs., 6 figs., 3 tabs.

  19. Identification of the functional elements in the promoter region of human DNA topoisomerase IIIbeta gene.

    PubMed

    Cho, Young Hoon; Park, Jee Young; Han, Sang Youp; Chung, In Kwon

    2004-09-17

    In this study, we have isolated and characterized the promoter region of the human DNA topoisomerase IIIbeta (hTOP3beta) gene. The 5' RACE assay showed a short exon 1 encoding only the 35-bp untranslated region and suggested the presence of multiple transcription initiation sites. The hTOP3beta gene promoter lacks a canonical TATA box or initiation element and is moderately high in GC content. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequence identified an activator element between -141 and -119 upstream of the transcription initiation site and a second regulatory element between -91 and -71. On the basis of scanning mutations of triple nucleotides, we demonstrated that a 5'GGAACC3' element between -117 and -112 plays a critical role in the up-regulation of the basal transcription activity. Changing the 5'GGAACC3' sequence leads to markedly reduced promoter activity. Gel mobility shift assays revealed that the 5'GGAACC3' element is required for DNA binding by the transcription factor complex. These observations lead to the conclusion that the positive regulatory region including the 5'GGAACC3' core element is essential for efficient expression of the hTOP3beta gene as well as for the binding of as yet unidentified regulatory factor(s).

  20. 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.

  1. 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.

  2. miR-214 regulates lactoferrin expression and pro-apoptotic function in mammary epithelial cells.

    PubMed

    Liao, Yalin; Du, Xiaogu; Lönnerdal, Bo

    2010-09-01

    Lactoferrin (Lf) is an abundantly expressed protein in human milk. Lactoferrin exhibits several important biological functions, and its expression is regulated by multiple environmental factors. Cellular endogenous factors, however, have not been extensively studied with regard to lactoferrin gene expression. In this study, we showed that lactoferrin gene expression and function are directly targeted by miR-214 in HC11 and MCF7 cells. In the lactoferrin mRNA 3 prime untranslated region (UTR) of human, mouse, rat, pig, bovine, camel, and goat species, there is a conserved region that perfectly matches the seed region of miR-214. Transfection of miR-214 mimic in HEK293 cells dose-dependently inhibited the activity of pGL3-control vector containing lactoferrin mRNA 3 prime UTR downstream of the luciferase gene. In HC11 cells, miR-214 overexpression inhibited the induction of lactoferrin expression by beta -estradiol (E2) and dexamethasone-prolactin-insulin (DPI). Furthermore, in MCF7 cells, overexpression of miR-214 markedly decreased lactoferrin expression (P lt 0.05), and inhibition of endogenous miR-214 expression increased lactoferrin expression and cellular apoptotic activities (P lt 0.05). In summary, our data showed that miR-214 is directly involved in lactoferrin expression and lactoferrin mediated cancer susceptibility (proapoptotic activities) in mammary epithelial cells.

  3. No association between schizophrenia and polymorphisms within the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT)

    SciTech Connect

    Daniels, J.; Williams, J.; Asherson, P.; McGuffin, P.; Owen, M.

    1995-02-27

    It has been suggested that the cytochrome P450 mono-oxygenase, debrisoquine 4-hydroxylase, is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells. It is also thought to be related to the dopamine transporter that acts to take released dopamine back up into presynaptic terminals. The present study used the association approach to test the hypothesis that mutations in the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT) confer susceptibility to schizophrenia. There were no differences in allele or genotype frequencies between patients and controls in the mutations causing the poor metaboliser phenotype in CYP2D6. In addition there was no association found between schizophrenia and a 48 bp repeat within the 3{prime} untranslated region of DAT. 18 refs., 2 tabs.

  4. NcoI and TaqI RFLPs for human M creatine kinase (CKM)

    SciTech Connect

    Perryman, M.B.; Hejtmancik, J.F.; Ashizawa, Tetsuo; Armstrong, R.; Lin, Sunchiang; Roberts, R.; Epstein, H.F. )

    1988-09-12

    Probe pHMCKUT contains a 135 bp cDNA fragment inserted into pGEM 3. The probe corresponds to nucleotides 1,201 to 1,336 located in the 3{prime} untranslated region of human M creatine kinase. The probe is specific for human M creatine kinase and does not hybridize to human B cretine kinase sequences. NcoI identifies a two allele polymorphism of a band at either 2.5 kb or 3.6 kb. TaqI identifies a two allele polymorphism at either 3.8 kb or 4.5 kb. Human M creatine has been localized to chromosome 19q. Autosomal co-dominant inheritance was shown in six informative Caucasian families.

  5. Aberrant splicing and transcription termination caused by P element insertion into the intron of a Drosophila gene

    SciTech Connect

    Horowitz, H.; Berg, C.A.

    1995-01-01

    Insertional mutagenesis screens using the P[lacZ, rosy{sup +}] (PZ) transposable element have provided thousands of mutant lines for analyzing genes of varied function in the fruitfly, Drosophila melanogaster. As has been observed with other P elements, many of the PZ-induced mutations result from insertion of the P element into the promoter or 5{prime} untranslated regions of the affected gene. We document here a novel mechanism for mutagenesis by this element. We show that sequences present within the element direct aberrant splicing and termination events that produce an mRNA composed of 5{prime} sequences from the mutated gene (in this case, pipsqueak) and 3{prime} sequences from within the P[lacZ, rosy{sup +}] element. These truncated RNAs could yield proteins with dominant mutant effects. 43 refs., 4 figs.

  6. Regional Sustainable Environmental Management

    EPA Science Inventory

    Regional sustainable environmental management is an interdisciplinary effort to develop a sufficient understanding of the interactions between ecosystems, the economy, law, and technology to formulate effective long-term management strategies on a regional scale. Regional sustai...

  7. Regional Sustainable Environmental Management

    EPA Science Inventory

    Regional sustainable environmental management is an interdisciplinary effort to develop a sufficient understanding of the interactions between ecosystems, the economy, law, and technology to formulate effective long-term management strategies on a regional scale. Regional sustai...

  8. Genome-Wide Analyses in Bacteria Show Small-RNA Enrichment for Long and Conserved Intergenic Regions

    PubMed Central

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Palumbo, Michael

    2014-01-01

    Interest in finding small RNAs (sRNAs) in bacteria has significantly increased in recent years due to their regulatory functions. Development of high-throughput methods and more sophisticated computational algorithms has allowed rapid identification of sRNA candidates in different species. However, given their various sizes (50 to 500 nucleotides [nt]) and their potential genomic locations in the 5′ and 3′ untranslated regions as well as in intergenic regions, identification and validation of true sRNAs have been challenging. In addition, the evolution of bacterial sRNAs across different species continues to be puzzling, given that they can exert similar functions with various sequences and structures. In this study, we analyzed the enrichment patterns of sRNAs in 13 well-annotated bacterial species using existing transcriptome and experimental data. All intergenic regions were analyzed by WU-BLAST to examine conservation levels relative to species within or outside their genus. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicate that sRNAs are enriched in intergenic regions, which are longer and more conserved than the average intergenic regions in the corresponding bacterial genome. We also found that sRNA-coding regions have different conservation levels relative to their flanking regions. This work provides a way to analyze how noncoding RNAs are distributed in bacterial genomes and also shows conserved features of intergenic regions that encode sRNAs. These results also provide insight into the functions of regions surrounding sRNAs and into optimization of RNA search algorithms. PMID:25313390

  9. Genome-wide analyses in bacteria show small-RNA enrichment for long and conserved intergenic regions.

    PubMed

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Palumbo, Michael; Contreras, Lydia M

    2015-01-01

    Interest in finding small RNAs (sRNAs) in bacteria has significantly increased in recent years due to their regulatory functions. Development of high-throughput methods and more sophisticated computational algorithms has allowed rapid identification of sRNA candidates in different species. However, given their various sizes (50 to 500 nucleotides [nt]) and their potential genomic locations in the 5' and 3' untranslated regions as well as in intergenic regions, identification and validation of true sRNAs have been challenging. In addition, the evolution of bacterial sRNAs across different species continues to be puzzling, given that they can exert similar functions with various sequences and structures. In this study, we analyzed the enrichment patterns of sRNAs in 13 well-annotated bacterial species using existing transcriptome and experimental data. All intergenic regions were analyzed by WU-BLAST to examine conservation levels relative to species within or outside their genus. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicate that sRNAs are enriched in intergenic regions, which are longer and more conserved than the average intergenic regions in the corresponding bacterial genome. We also found that sRNA-coding regions have different conservation levels relative to their flanking regions. This work provides a way to analyze how noncoding RNAs are distributed in bacterial genomes and also shows conserved features of intergenic regions that encode sRNAs. These results also provide insight into the functions of regions surrounding sRNAs and into optimization of RNA search algorithms.

  10. Nucleotide sequence of the 3'-terminal region of potato virus YN RNA.

    PubMed

    van der Vlugt, R; Allefs, S; de Haan, P; Goldbach, R

    1989-01-01

    The sequence of the 3'-terminal 1611 nucleotides of the genome of the tobacco veinal necrosis strain of potato virus Y (PVYN) was determined. The sequence revealed an open reading frame of 1285 nucleotides, of which the start was not identified, and an untranslated region of 316 nucleotides upstream of a poly(A) tract. Comparison of the open reading frame with the amino-terminal sequence of the viral coat protein enabled mapping of the start of the coat protein at amino acid -267, and indicated that maturation of this protein requires proteolytic processing from a larger polyprotein precursor at a glutamine/glycine dipeptide sequence. The coat protein of PVYN displayed significant (51 to 63%) sequence homology to the coat proteins of four other potyviruses, tobacco etch virus, tobacco vein mottling virus, plum pox virus and sugarcane mosaic virus. Even higher sequence homology (91%) was detected with the coat protein of a fifth potyvirus, pepper mottle virus (PeMV). This homology was of the same level as found between the coat proteins of PVYN and a second strain of this virus, PVYD. Since, moreover, PVYN and PeMV were the only potyviruses displaying homology in the 3'-terminal, non-translated regions of their genomes, we conclude that PeMV should be regarded as a strain of PVY.

  11. Regional differences in the genetic variability of Finno-Ugric speaking Komi populations.

    PubMed

    Khrunin, Andrey; Verbenko, Dmitry; Nikitina, Kseniya; Limborska, Svetlana

    2007-01-01

    The Komi (Komi-Zyryan) people are one of the most numerous ethnic groups belonging to the Finno-Ugric linguistic community. They occupy an extensive territory in north Russia to the west of the Ural Mountains, in the northeast of the East European Plain. This is an area of long-term interactions between Europeans and North Asians. Genetic variability was evaluated in two geographically distinct populations, the Izhemski and Priluzski Komi. We searched for polymorphisms of the TP53 gene (a 16-bp duplication in intron 3 and three RFLPs: for Bsh1236I at codon 72, for MspI in intron 6, and for BamHI in the 3' flanking region) and for variable number tandem repeat (VNTR) polymorphisms of locus D1S80 and of the 3' untranslated region of the gene for apolipoprotein B (ApoB). Some data from our previous studies of TP53, 3'ApoB, and D1S80 variability were involved in the comparison of Komi with other Eastern European populations. Multidimensional scaling analysis of genetic distances was used for the evaluation of genetic relationships between populations. The results revealed some affinity between Priluzski Komi and Eastern Slavonic populations, and significant segregation of Izhemski Komi from other ethnic groups studied. The unique genetic features of Izhemski Komi may have been determined by their ethnogenesis or the pressure of environmental factors, such as special nutrition and adaptation to extreme climatic conditions.

  12. The leader region of Laminin B1 mRNA confers cap-independent translation.

    PubMed

    Petz, Michaela; Kozina, Daniela; Huber, Heidemarie; Siwiec, Tanja; Seipelt, Joachim; Sommergruber, Wolfgang; Mikulits, Wolfgang

    2007-01-01

    Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5'-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5'-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes.

  13. 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

  14. Analysis of the promoter region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

    PubMed Central

    Bélanger, C; Peri, K G; MacKenzie, R E

    1991-01-01

    Sequence analysis of the 5'-flanking region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) revealed several putative cis-regulatory elements. To delineate the function of these regulatory elements, various deletion mutants of the 5'-flanking region were connected to the reporter gene chloramphenicol acetyltransferase (CAT) and promoter activity was measured in transient transfection assays. Transfection experiments performed with the sequence extending from -508 to +59 produced a high-level transient expression of the CAT gene in BALB/c 3T3-SV-T2 and NIH 3T3 cells. Removal of the sequence from +16 to +59 which includes the second transcription start point at +43, a TATA-like box and 5'-untranslated sequences abolished the promoter activity. Deletion analysis of 5'-upstream sequences revealed that the region from positions -55 to +59 is sufficient to mediate a high CAT activity comparable to the level obtained with the construct -508/+59. Within this region are found a CAAT box, a TATA-like box and two putative GC boxes. A functional analysis of the promoter showed that the sequence from -55 to +59 is sufficient to respond to stimulation by serum. Images PMID:1843253

  15. Characterization of the 5' region of the Leishmania infantum LORIEN/MAT2 gene cluster and role of LORIEN flanking regions in post-transcriptional regulation.

    PubMed

    García-Estrada, Carlos; Pérez-Pertejo, Yolanda; Ordóñez, David; Balaña-Fouce, Rafael; Reguera, Rosa M

    2008-09-01

    LORIEN (encoding a protein that contains a SP-RING/Miz zinc-finger motif present in a group of proteins involved in the Small Ubiquitin-related Modifier -SUMO- conjugation pathway) and MAT2 (encoding the methionine adenosyltransferase -MAT-) genes are arranged as two alternating copies in a head-to-tail configuration, with the LORIEN gene as the first copy of the cluster. The 5880bp preceding the first LORIEN gene copy were compared to the same region of L. major, showing a 93% identity between them. Bioinformatic analysis of this region predicted the presence of a 747-bp ORF encoding a hypothetical protein of 248 amino acids. Transcription of this ORF was confirmed by run-on assays and RT-PCR. Expression of the LORIEN gene was tested in both the promastigote and amastigote stages. Transcription arrest evidenced that LORIEN mRNA stability was very similar in both stages of the parasite life cycle. Protein synthesis inhibition by cycloheximide led to an increase in the steady-state levels of LORIEN transcripts only during the promastigote stage, pointing out to the existence of different stage-dependent mechanisms operating on the post-transcriptional regulation of this gene. The role of the LORIEN untranslated regions (5'UTR and 3'UTR) in post-transcriptional regulation was analysed using the luciferase (luc) reporter gene. Results evidenced that the 5'UTR was responsible for a low reporter gene expression, whereas the intergenic region (IR) between LORIEN and MAT2 genes provided high luc levels. However, the 3'UTR seemed to lack regulatory elements. Basing on these results, a model of regulation for the LORIEN gene is proposed.

  16. Mutations in noncoding regions of GJB1 are a major cause of X-linked CMT

    PubMed Central

    Tomaselli, Pedro J.; Rossor, Alexander M.; Horga, Alejandro; Jaunmuktane, Zane; Carr, Aisling; Saveri, Paola; Piscosquito, Giuseppe; Pareyson, Davide; Laura, Matilde; Blake, Julian C.; Poh, Roy; Polke, James; Houlden, Henry

    2017-01-01

    Objective: To determine the prevalence and clinical and genetic characteristics of patients with X-linked Charcot-Marie-Tooth disease (CMT) due to mutations in noncoding regions of the gap junction β-1 gene (GJB1). Methods: Mutations were identified by bidirectional Sanger sequence analysis of the 595 bases of the upstream promoter region, and 25 bases of the 3′ untranslated region (UTR) sequence in patients in whom mutations in the coding region had been excluded. Clinical and neurophysiologic data were retrospectively collected. Results: Five mutations were detected in 25 individuals from 10 kindreds representing 11.4% of all cases of CMTX1 diagnosed in our neurogenetics laboratory between 1996 and 2016. Four pathogenic mutations, c.-17G>A, c.-17+1G>T, c.-103C>T, and c.-146-90_146-89insT were detected in the 5′UTR. A novel mutation, c.*15C>T, was detected in the 3′ UTR of GJB1 in 2 unrelated families with CMTX1 and is the first pathogenic mutation in the 3′UTR of any myelin-associated CMT gene. Mutations segregated with the phenotype, were at sites predicted to be pathogenic, and were not present in the normal population. Conclusions: Mutations in noncoding DNA are a major cause of CMTX1 and highlight the importance of mutations in noncoding DNA in human disease. Next-generation sequencing platforms for use in inherited neuropathy should therefore include coverage of these regions. PMID:28283593

  17. miR-148 targets human DNMT3b protein coding region.

    PubMed

    Duursma, Anja M; Kedde, Martijn; Schrier, Mariette; le Sage, Carlos; Agami, Reuven

    2008-05-01

    MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.

  18. Mutations in noncoding regions of GJB1 are a major cause of X-linked CMT.

    PubMed

    Tomaselli, Pedro J; Rossor, Alexander M; Horga, Alejandro; Jaunmuktane, Zane; Carr, Aisling; Saveri, Paola; Piscosquito, Giuseppe; Pareyson, Davide; Laura, Matilde; Blake, Julian C; Poh, Roy; Polke, James; Houlden, Henry; Reilly, Mary M

    2017-04-11

    To determine the prevalence and clinical and genetic characteristics of patients with X-linked Charcot-Marie-Tooth disease (CMT) due to mutations in noncoding regions of the gap junction β-1 gene (GJB1). Mutations were identified by bidirectional Sanger sequence analysis of the 595 bases of the upstream promoter region, and 25 bases of the 3' untranslated region (UTR) sequence in patients in whom mutations in the coding region had been excluded. Clinical and neurophysiologic data were retrospectively collected. Five mutations were detected in 25 individuals from 10 kindreds representing 11.4% of all cases of CMTX1 diagnosed in our neurogenetics laboratory between 1996 and 2016. Four pathogenic mutations, c.-17G>A, c.-17+1G>T, c.-103C>T, and c.-146-90_146-89insT were detected in the 5'UTR. A novel mutation, c.*15C>T, was detected in the 3' UTR of GJB1 in 2 unrelated families with CMTX1 and is the first pathogenic mutation in the 3'UTR of any myelin-associated CMT gene. Mutations segregated with the phenotype, were at sites predicted to be pathogenic, and were not present in the normal population. Mutations in noncoding DNA are a major cause of CMTX1 and highlight the importance of mutations in noncoding DNA in human disease. Next-generation sequencing platforms for use in inherited neuropathy should therefore include coverage of these regions. Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

  19. Identification of brain-derived neurotrophic factor promoter regions mediating tissue-specific, axotomy-, and neuronal activity-induced expression in transgenic mice

    PubMed Central

    1995-01-01

    The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner. PMID:7822414

  20. Expression of adenovirus-2 early region 4: assignment of the early region 4 polypeptides to their respective mRNAs, using in vitro translation.

    PubMed

    Tigges, M A; Raskas, H J

    1982-12-01

    Adenovirus-2 early region 4 (E4; map positions 91.3 to 99.1) encodes six 5' and 3' coterminal, differently spliced mRNAs, which are 2.5, 2.1, 1.8, 1.5, 1.2, and 0.8 kilobases (kb) long. Hybridization selection with five cloned viral DNA fragments that hybridize with subsets of E4 mRNAs was used to purify these six mRNAs and a previously unreported 3.0-kb mRNA from virus-infected cells. E4 mRNAs which were purified by hybridization selection with cloned EcoRI fragment C (map positions 89.7 to 100) were also fractionated by size. The purified mRNAs were then translated in rabbit reticulocyte or wheat germ lysate systems. The full complement of E4 mRNAs specified as many as 16 different polypeptides, with molecular weights ranging from 24,000 (24K) to 10K. The most abundant E4 mRNA, which was 2.1 kb long, specified an 11K polypeptide. The 1.5-kb mRNA, which differed from the 2.1-kb mRNA only by deletion of a second intron from the 3' untranslated region, also specified an 11K polypeptide. The second most abundant mRNA, which was 1.8 kb long, and the 1.2-kb mRNA, which had an intron deleted from the 3' untranslated region, specified a 15K polypeptide. This polypeptide was labeled more intensely with [5,6-(3)H]leucine than with [35S]methionine. The 3.0- and 2.5-kb mRNAs specified four polypeptides (24K, 22K, 19K, and 17K). Translation of E4 mRNAs with a mean size of 0.8 kb, which accumulated preferentially in the presence of cycloheximide, yielded at least 10 polypeptides that migrated in polyacrylamide gels with apparent molecular weights ranging from 21,800 to 10,000. On the basis of translation in wheat germ lysates and the distribution of polypeptides encoded by size-fractionated mRNAs, we concluded that the 0.8-kb mRNA size class includes a heterogeneous mixture of mRNAs which are probably formed as the result of utilization of alternate splice acceptor and donor sites during removal of the second intron. Our polypeptide assignments for the 2.1-, 1.8-, 1.5-, and 1

  1. Epidermal surface antigen (MS17S1) is highly conserved between mouse and human

    SciTech Connect

    Cho, Y.J.; Chema, D.; Cho, M.

    1995-05-20

    A mouse monoclonal antibody ECS-1 raised to human keratinocytes detects a 35-kDa epidermal surface antigen (ESA) and causes keratinocyte dissociation in vitro. ECS-1 stains skin of 16-day mouse embryo and 8- to 9-week human fetus. Mouse Esa cDNA encodes a 379-amino-acid protein that is 99.2% identical to the human, differing at only 3 amino acids. The gene (M17S1) was mapped to mouse chromosome 11, highlighting the conserved linkage synteny existing between human chromosome 17 and mouse chromosome 11. Although the nude locus has been mapped to the same region of chromosome 11, no abnormalities in protein, mRNA, or cDNA or genomic sequences were detected in nude mice. However, both nude and control mice were found to have a second Esa mRNA transcript that conserves amino acid sequence and molecular weight. The mouse and human 5{prime} and 3{prime} untranslated sequences are conserved. Similar RNA folding patterns of the 5{prime} untranslated region are predicted despite a 91-bp insertion in the mouse. These data suggest that both the function and the regulation of ESA protein are of importance and that Esa (M17S1) is not the nude locus gene. 42 refs., 7 figs., 3 tabs.

  2. Colonial Museums in the Us (Un)translated

    ERIC Educational Resources Information Center

    Valdeón, Roberto A.

    2015-01-01

    This paper examines the role of museums in the creation of anglophone stories in the USA, and how the (non-)translation of signs contributes to create a narrative of exclusion vis-à-vis other groups, notably native Americans, the Spanish, and the French. Particular attention is paid to open-air museums that preserve old buildings and areas…

  3. Colonial Museums in the Us (Un)translated

    ERIC Educational Resources Information Center

    Valdeón, Roberto A.

    2015-01-01

    This paper examines the role of museums in the creation of anglophone stories in the USA, and how the (non-)translation of signs contributes to create a narrative of exclusion vis-à-vis other groups, notably native Americans, the Spanish, and the French. Particular attention is paid to open-air museums that preserve old buildings and areas…

  4. Search for the Untranslated, Bibliographic Research by Translators.

    ERIC Educational Resources Information Center

    Jasenas, Michael

    1984-01-01

    Presents a guide of basic bibliographical sources of English translations in the humanities and the social sciences. The sources fall into the following categories: (1) general bibliographies of translations, (2) humanities, (3) social sciences, (4) national bibliographies, and (5) library catalogs. (SED)

  5. Characterization of direct selected cDNAs from the BRCA1 region of 17q21

    SciTech Connect

    Welcsh, P.L.; Osborne-Lawrence, S.L.; Spillman, M.A.

    1994-09-01

    A gene involved in the development of early-onset familial breast and ovarian cancer, BRCA1, has been mapped to human chromosome 17q21. Polymorphisms closely linked to BRCA1 has been sublocalized to a region of 17q21 which is defined by the markers D17S856 and D17S78. A physical map of this region, that consists of yeast artificial chromosome (YAC) and cosmid contigs, has been constructed and used to isolate potential coding sequences via direct selection. We have identified at least 23 unique transcripts in a 600 kb interval corresponding to approximately one gene every 30 kb. We have determined the expression profile of these cDNAs by generating cDNA-specific primers which have been used in a screen of cDNAs derived from wide variety of tissues and cell types. Full length cDNA clones are being obtained from cDNA libraries in which the genes have been shown to be expressed by a variety of techniques which include direct screening, 5{prime} and 3{prime} RACE, anchor PCR as well as modified selection procedures. We are currently screening for mutations in these candidate cDNAs in affected family members known to harbor a germ-line BRCA1 mutation and in sporadic breast and ovarian tumors. Mutation screening is being performed by Southern and Northern blotting, DNA sequencing, and SSCP analysis of germline DNA and cDNA. Finally, we are analyzing these candidate cDNAs in a number of breast and ovarian cancer cell lines for induction by known mitogenic factors such as estrogen and progesterone by Northern blotting and RT-PCR.

  6. Construction of a genomic library of the human cytomegalovirus genome and analysis of late transcription of its inverted internal repeat region

    SciTech Connect

    Silva, K.F.S.T.

    1989-01-01

    The investigations described in this dissertation were designed to determine the transcriptionally active DNA sequences of IIR region and to identify the viral mRNA transcribed from the transcriptionally most active DNA sequences of that region during late phase of HCMV Towne infection. Preliminary transcriptional studies which included the hybridization of a southern blot of XbaI digested entire HCMV genome to {sup 32}P-labelled late phase infected cell A{sup +} RNA, indicated that late viral transcripts homologous to XbaI Q fragment of IIR region were very highly abundant while XbaI Q fragment showed a very low transcriptional activity. To facilitate further analysis of late transcription of IIR region, the entire DNA sequences of IIR region were molecularly cloned as U, S, and H BamHI fragments in pACYC-184 plasmid vector. In addition, to be used in future studies on other regions of the genome, except for y and c{prime} smaller fragments the entire 240 kb HCMV genome was cloned as BamHI fragments in the same vector. Furthermore, the U, S, and H BamHI fragments were mapped with six other restriction enzymes in order to use that mapping data in subsequent transcriptional analysis of the IIR region. Further localization of transcriptionally active DNA sequences within IIR region was achieved by hybridization of southern blots of restricted U, S, and H BamHI fragments with 3{prime} {sup 32}P-labelled infected cell late A{sup +} RNA. The 1.5 kb EcooRI subfragments of S BamHI fragment and the adjoining 0.72 kb XhoI subfragment of H BamHI fragment revealed the highest level of transcription, although the remainder of the S fragment was also transcribed at a substantial level. The U fragment and the remainder of the H fragment was transcribed at a very low level.

  7. Regional Smart Growth Alliances

    EPA Pesticide Factsheets

    This page describes the Urban Land Institute regional smart growth alliances that received funding from EPA to help support economic development, accommodate growth, enhance quality of, and protect the environment in regions across the country.

  8. Region 9 Tribal Program

    EPA Pesticide Factsheets

    EPA Region 9 helps tribes strengthen management of environmental programs in Indian country, and ensure that tribes have a voice in decisions. The region serves 148 federally recognized tribes with the states of Arizona, California, and Nevada.

  9. ERLN Regional Support

    EPA Pesticide Factsheets

    Regional labs play important roles in the Environmental Response Laboratory Network. They can serve as point of contact; coordinate sample flow, special analytical service requests, or training exercises; and partner with regional emergency/disaster staff.

  10. Region 9 RTOC Charter

    EPA Pesticide Factsheets

    U.S. EPA Pacific Southwest (Region 9) Regional Tribal Operations Committee (RTOC) Charter as amended 11/13/2014: Mission, Goals, Scope, Structure & Membership, Meetings, Administration, Charter Amendment/Review, and Current Working Draft.

  11. [Regional aging in Germany].

    PubMed

    Bucher, H

    1996-01-01

    Elderly people in Germany have a specific regional distribution. Recent regional population projections show that these patterns will change. The most dynamic process of aging will take place in the suburban parts of the large western Germany agglomerations, whereas in eastern Germany aging concentrates in regions with a lower density. There will be a regional deconcentration of elderly people with consequences for the planning of infrastructure.

  12. Learning Regions in Germany

    ERIC Educational Resources Information Center

    Thinesse-Demel, Jutta

    2010-01-01

    In 2000, the German Federal Ministry of Education and Research (BMBF) launched the programme "Learning Regions--Providing Support for Networks'" in cooperation with the Lander. It was co-financed by the European Social Fund (ESF). Some 90 regions were selected and financially supported. After one year, 71 regions continued to build-up…

  13. Learning Regions in Germany

    ERIC Educational Resources Information Center

    Thinesse-Demel, Jutta

    2010-01-01

    In 2000, the German Federal Ministry of Education and Research (BMBF) launched the programme "Learning Regions--Providing Support for Networks'" in cooperation with the Lander. It was co-financed by the European Social Fund (ESF). Some 90 regions were selected and financially supported. After one year, 71 regions continued to build-up…

  14. HRM: HII Region Models

    NASA Astrophysics Data System (ADS)

    Wenger, Trey V.; Kepley, Amanda K.; Balser, Dana S.

    2017-07-01

    HII Region Models fits HII region models to observed radio recombination line and radio continuum data. The algorithm includes the calculations of departure coefficients to correct for non-LTE effects. HII Region Models has been used to model star formation in the nucleus of IC 342.

  15. Convergent transcription initiates from oppositely oriented promoters within the 5 prime end regions of Drosophila melanogaster F elements

    SciTech Connect

    Minchiotti, G. ); Di Nocera, P.P. )

    1991-10-01

    Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that likely propagate by the retrotranscription of RNA intermediates. Plasmids bearing DNA segments from the left-hand region of a full-length F element fused to the CAT gene were used as templates for transient expression assays in Drosophila Schneider II cultured cells. Protein and RNA analyses led to the identification of two promoters, F{sub in} and F{sub out}, that transcribe in opposite orientations. Analysis of the template activity of 3{prime} deletion derivatives indicates that the level of accumulation of F{sub in}RNA is also dependent upon the presence of sequences located within the +175 to +218 interval. The F{sub out} promoter drives transcription in the opposite orientation with respect to F{sub in}, F{sub out} transcripts initiate at nearby sites within the +92 to +102 interval. Sequences downstream of these multiple RNA start sites are not required for the activity of the F{sub out} promoter. Deletions knocking out the F{sub in} promoter do not impair F{sub out} transcription; conversely, initiation at the F{sub in} promoter still takes place in templates that lack the F{sub out} promoter. At a low level, both promoters are active in cultured cells.

  16. Fine mapping and narrowing of the genetic interval of the spinal muscular atrophy region by linkage studies

    SciTech Connect

    Wirth, B.; Voosen, B.; Roehrig, D.; Piechaczek, B.; Ruonk-Schoeneborn, S.; Zerres, K. ); Knapp, M. )

    1993-01-01

    The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped to chromosome 5q12.2-q13, within a genetic distance of about 6 cM, and is proximally flanked by the locus D5S6 and distally by D5S112. Here, we report linkage analyses in 64 SMA families with nine polymorphic markers closely linked to the SMA gene which allowed us to narrow the SMA region to about 4cM and to define a new proximal genetic border by the locus D5S125 EF(TG/AG)[sub n]. B