Science.gov

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. Structural organization of the human. alpha. -galactosidase A gene: Further evidence for the absence of a 3 prime untranslated region

    SciTech Connect

    Bishop, D.F.; Kornreich, R.; Desnick, R.J. )

    1988-06-01

    Human {alpha}-galactosidase A is a lysosomal hydrolase encoded by a gene localized to the chromosomal region Xq22. The deficient activity of this enzyme results in Fabry disease, an X chromosome-linked recessive disorder that leads to premature death in affected males. For studies of the structure and function of {alpha}-galactosidase A and for characterization of the genetic lesions in families with Fabry disease, the full-length cDNA was isolated, sequenced, and used to screen human genomic libraries. The 1393-base-pair full-length cDNA had a 60-nucleotide 5{prime} untranslated region and encoded a precursor peptide of 429 amino acids including a signal peptide of 31 residues. Three overlapping {lambda} clones spanning 32 kilobases were identified that contained the entire {approx}12-kilobase chromosomal gene as well as {approx}9 and {approx}11 kilobases of 5{prime} and 3{prime} flanking sequence, respectively. The gene had seven exons. The genomic exonic and full-length cDNA sequences were identical. There was an upstream HTF island (Hpa II tiny fragments) followed by four direct repeats of the chorion box enhancer. The unique lace of a 3{prime} untranslated sequence in the {alpha}-galactosidase A cDNA was confirmed by sequencing additional cDNA clones and the genomic 3{prime} region.

  3. 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.

  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. 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.

  6. 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. PMID:27262928

  7. 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.

  8. 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

  9. Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs.

    PubMed

    Mendrysa, S M; McElwee, M K; Perry, M E

    2001-02-01

    The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.

  10. Secondary structure of the 3' untranslated region of flaviviruses: similarities and differences.

    PubMed

    Proutski, V; Gould, E A; Holmes, E C

    1997-03-15

    Genetic algorithm-based RNA secondary structure prediction was used in combination with comparative sequence analysis to construct models of folding for the distal part of the 3'-untranslated region of flaviviruses belonging to four serological groups. Elements of RNA secondary structure that are preserved among all the flaviviruses studied were revealed, despite the high degree of sequence divergence between them. At the same time, structural elements were observed that distinguish members of different serological groups and, in particular, a region of remarkable structural divergence between the tick-borne and mosquito-borne flaviviruses was found. Application of the genetic algorithm also revealed that the 3'-terminus of flaviviral genomic RNA may take on alternative conformations, which are not observed in the 3'-terminus of complementary minus strand RNA. These alternative folding patterns may have roles in the regulation of transcription and translation initiation and in the switch between them.

  11. Cloning and polymorphisms of the 3' untranslated region of malic enzyme gene in Chinese red cattle.

    PubMed

    Zhou, G L; Zhang, G L; Cao, Y; Jin, H G

    2011-09-01

    The objective of this study was to identify alternative transcripts and single nucleotide polymorphisms (SNPs) in the 3'-untranslated region (3' UTR) of bovine malic enzyme (ME1) gene and to evaluate the extent to which polymorphisms were associated with meat quality and carcass traits in Chinese red cattle. Two transcripts, long transcript and short transcript that differ in the length of the 3' UTR were cloned. A single nucleotide polymorphism was detected in 3' UTR and a restriction site for endonuclease ME1-Dra I was also found. The result revealed that the ME1-Dra I genotypes had a significant effect on cooking loss, pH measured 24h post-mortem (pH(24h)) and eye muscle area (P < 0.05). In conclusion, the SNPs may be used as DNA markers to select for meat quality and carcass traits in Chinese red cattle.

  12. Secondary structure models of the 3′ untranslated regions of diverse R2 RNAs

    PubMed Central

    RUSCHAK, AMY M.; MATHEWS, DAVID H.; BIBILLO, ARKADIUSZ; SPINELLI, SHERRY L.; CHILDS, JESSICA L.; EICKBUSH, THOMAS H.; TURNER, DOUGLAS H.

    2004-01-01

    The RNA structure of the 3′ untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure–function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3′ UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3′ UTR R2 RNA of Forficula auricularia. The predicted structures for Bombyx mori and F. auricularia are consistent with chemical modification data obtained with β-ethoxy-α-ketobutyraldehyde (kethoxal), dimethyl sulfate, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. The structures appear to have common helices that are likely important for function. PMID:15146081

  13. Translational control by 5'-untranslated regions of eukaryotic mRNAs.

    PubMed

    Hinnebusch, Alan G; Ivanov, Ivaylo P; Sonenberg, Nahum

    2016-06-17

    The eukaryotic 5' untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5' upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5'UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer.

  14. Translational control by 5'-untranslated regions of eukaryotic mRNAs.

    PubMed

    Hinnebusch, Alan G; Ivanov, Ivaylo P; Sonenberg, Nahum

    2016-06-17

    The eukaryotic 5' untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5' upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5'UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer. PMID:27313038

  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. Influence of Theiler's murine encephalomyelitis virus 5' untranslated region on translation and neurovirulence.

    PubMed

    Stein, S B; Zhang, L; Roos, R P

    1992-07-01

    The DA strain of Theiler's murine encephalomyelitis virus (TMEV), a picornavirus, causes a persistent, restricted infection and demyelinating disease in mice. In contrast, the GDVII strain causes an acute, fatal, neuronal disease and is highly neurovirulent. To investigate the role of the TMEV 5' untranslated region (UTR) in translational efficiency and the TMEV subgroup differences, we tested the translational efficiency of transcripts in vitro derived from plasmids containing DA, GDVII, or DA/GDVII chimeric 5' UTRs preceding a reporter gene or the rest of the TMEV genome. We demonstrated that GDVII RNA translates more efficiently in rabbit reticulocyte lysate than DA RNA and that this enhanced translation is mediated by multiple domains in the GDVII 5' UTR as well as a region of the genome outside of the 5' UTR. We also identified a region within DA nucleotides 14 to 395 which inhibits translation of DA RNA and could contribute to the persistent, restricted DA central nervous system infection; the predicted secondary structure of the 5' UTR demonstrates a remarkable stem-loop structure within this region that is relatively unique among picornaviruses. Data from experiments involving DA/GDVII chimeric 5' UTR full-length infectious cDNA clones suggested that sequences in the 5' UTR can affect the neurovirulence phenotype but that translational efficiency is necessary but not sufficient for neurovirulence. These studies emphasize the multigenic nature of neurovirulence and the importance of translation in the regulation of picornaviral gene expression.

  17. Influence of Theiler's murine encephalomyelitis virus 5' untranslated region on translation and neurovirulence.

    PubMed Central

    Stein, S B; Zhang, L; Roos, R P

    1992-01-01

    The DA strain of Theiler's murine encephalomyelitis virus (TMEV), a picornavirus, causes a persistent, restricted infection and demyelinating disease in mice. In contrast, the GDVII strain causes an acute, fatal, neuronal disease and is highly neurovirulent. To investigate the role of the TMEV 5' untranslated region (UTR) in translational efficiency and the TMEV subgroup differences, we tested the translational efficiency of transcripts in vitro derived from plasmids containing DA, GDVII, or DA/GDVII chimeric 5' UTRs preceding a reporter gene or the rest of the TMEV genome. We demonstrated that GDVII RNA translates more efficiently in rabbit reticulocyte lysate than DA RNA and that this enhanced translation is mediated by multiple domains in the GDVII 5' UTR as well as a region of the genome outside of the 5' UTR. We also identified a region within DA nucleotides 14 to 395 which inhibits translation of DA RNA and could contribute to the persistent, restricted DA central nervous system infection; the predicted secondary structure of the 5' UTR demonstrates a remarkable stem-loop structure within this region that is relatively unique among picornaviruses. Data from experiments involving DA/GDVII chimeric 5' UTR full-length infectious cDNA clones suggested that sequences in the 5' UTR can affect the neurovirulence phenotype but that translational efficiency is necessary but not sufficient for neurovirulence. These studies emphasize the multigenic nature of neurovirulence and the importance of translation in the regulation of picornaviral gene expression. Images PMID:1602556

  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. An essential secondary structure in the 3' untranslated region of the mouse hepatitis virus genome.

    PubMed

    Hsue, B; Masters, P S

    1998-01-01

    The 3' untranslated regions (3' UTRs) of coronaviruses contain the signals necessary for negative strand RNA synthesis and may also harbor elements essential for positive strand replication and subgenomic RNA transcription. The 3' UTRs of mouse hepatitis virus (MHV) and bovine coronavirus (BCV) are more than 30% divergent. In an effort to learn what parts of these regions might be functionally interchangeable, we attempted to replace the 3' UTR of MHV with its BCV counterpart by targeted RNA recombination. Initially, we tried to substitute the 3' 267 nucleotides (nt) of the 301 nt MHV 3' UTR with the corresponding region of the BCV 3' UTR. This exchange did not yield viable recombinant viruses, and the donor DI RNA was shown to be unable to replicate with MHV as a helper virus. Subsequent analysis revealed that the entire BCV 3' UTR could be inserted into the MHV genome in place of the entire MHV 3' UTR. It resulted that the failure of the initial attempted substitution was due to the inadvertent disruption of an essential conserved bulged stem-loop secondary structure in the MHV and BCV 3' URTs immediately downstream of the N gene stop codon.

  20. Cap-independent translation by the 5' untranslated region of Theiler's murine encephalomyelitis virus.

    PubMed Central

    Bandyopadhyay, P K; Wang, C; Lipton, H L

    1992-01-01

    The RNA genome of Theiler's murine encephalomyelitis viruses, a picornavirus belonging to the genus Cardiovirus, is translated in infected cells to a polyprotein. Unlike cellular messages, the 5' end of the RNA is not capped, and the untranslated region (UTR) is quite long (1,064 nucleotides in size). In poliovirus and encephalomyocarditis virus, the 5'UTR is thought to mediate cap-independent translation. We report here experiments to determine the role of the Theiler's murine encephalomyelitis virus 5'UTR in translation. Recombinant DNAs were constructed that were transcribed into bicistronic mRNAs encoding 5' chloramphenicol acetyltransferase intercistronic sequences linked to luciferase and a poly(A) 3' tail. The sequences of the 5'UTR, either complete or with sequential 5' deletions, were inserted into the intercistronic region. Bicistronic RNA transcripts were translated in a rabbit reticulocyte lysate or used to transfect BHK-21 cells, and chloramphenicol acetyltransferase and luciferase synthesis was quantitated. The results strongly suggest that the Theiler's virus 5'UTR promotes cap-independent translation and that the 5' boundary of the relevant signals resides 3' to nucleotide 500. Monocistronic mRNAs were synthesized by using an expression vector in which the 5'UTR containing deletions at the 3' terminus was inserted 5' to the coding sequences for luciferase. Analysis of luciferase translation in a rabbit reticulocyte lysate suggests that the 3' end of the translation initiation signal lies between nucleotides 1043 and 1053. Images PMID:1404591

  1. Cap-independent translation by the 5' untranslated region of Theiler's murine encephalomyelitis virus.

    PubMed

    Bandyopadhyay, P K; Wang, C; Lipton, H L

    1992-11-01

    The RNA genome of Theiler's murine encephalomyelitis viruses, a picornavirus belonging to the genus Cardiovirus, is translated in infected cells to a polyprotein. Unlike cellular messages, the 5' end of the RNA is not capped, and the untranslated region (UTR) is quite long (1,064 nucleotides in size). In poliovirus and encephalomyocarditis virus, the 5'UTR is thought to mediate cap-independent translation. We report here experiments to determine the role of the Theiler's murine encephalomyelitis virus 5'UTR in translation. Recombinant DNAs were constructed that were transcribed into bicistronic mRNAs encoding 5' chloramphenicol acetyltransferase intercistronic sequences linked to luciferase and a poly(A) 3' tail. The sequences of the 5'UTR, either complete or with sequential 5' deletions, were inserted into the intercistronic region. Bicistronic RNA transcripts were translated in a rabbit reticulocyte lysate or used to transfect BHK-21 cells, and chloramphenicol acetyltransferase and luciferase synthesis was quantitated. The results strongly suggest that the Theiler's virus 5'UTR promotes cap-independent translation and that the 5' boundary of the relevant signals resides 3' to nucleotide 500. Monocistronic mRNAs were synthesized by using an expression vector in which the 5'UTR containing deletions at the 3' terminus was inserted 5' to the coding sequences for luciferase. Analysis of luciferase translation in a rabbit reticulocyte lysate suggests that the 3' end of the translation initiation signal lies between nucleotides 1043 and 1053.

  2. 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. PMID:17719098

  3. 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.

  4. Characterization of the Human Ornithine Transcarbamylase 3′ Untranslated Regulatory Region

    PubMed Central

    Lopes-Marques, Monica; Pereira-Castro, Isabel; Amorim, António

    2012-01-01

    Mutations in the untranslated regulatory regions of genes may result in abnormal gene expression or transcriptional regulation. In this study, we characterize the ornithine transcarbamylase (OTC) mRNA isoforms of the X-linked OTC gene involved in the urea formation in the liver. Our data revealed that two major transcripts (OTC-t1 and OTC-t2) are more highly expressed than any of the other isoforms in all the tissues analyzed, though a longer transcript (OTC-t3) was also isolated and characterized from the brain sample. The OTC-t2 sequence fully matches the OTC mRNA reference sequence (NM_000531.5). All three isoforms use a canonical AAUAAA hexamer that is predicted to fold into a hairpin secondary structure which might be exposed to the cleavage and polyadenylation specificity factor. In addition, we observed that the OTC-t1 and OTC-t2 transcripts display heterogeneity at the cleavage sites in a tissue-dependent manner. Taken together, our data demonstrate that several mRNA isoforms are transcribed from the OTC gene, thereby indicating a wide degree of variability in post-transcriptional regulation. PMID:22054066

  5. Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences.

    PubMed

    Hahn, C S; Hahn, Y S; Rice, C M; Lee, E; Dalgarno, L; Strauss, E G; Strauss, J H

    1987-11-01

    We have isolated a cDNA clone after reverse transcription of the genomic RNA of Asibi yellow fever virus whose structure suggests it was formed by self-priming from a 3'-terminal hairpin of 87 nucleotides in the genomic RNA. We have also isolated a clone from cDNA made to Murray Valley encephalitis virus RNA that also appears to have arisen by self-priming from a 3'-terminal structure very similar or identical to that of yellow fever. In addition, 3'-terminal sequencing of the S1 strain of dengue 2 RNA shows that this RNA is also capable of forming a 3'-terminal hairpin of 79 nucleotides. Furthermore, we have identified two 20-nucleotide sequence elements which are present in the 3' untranslated region of all three viruses; one of these sequence elements is repeated in Murray Valley encephalitis and dengue 2 RNA but not in yellow fever RNA. In all three viruses, which represent the three major serological subgroups of the mosquito-borne flaviviruses, the 3'-proximal conserved sequence element, which is found immediately adjacent to the potential 3'-terminal hairpin, is complementary to another conserved domain near the 5' end of the viral RNAs, suggesting that flavivirus RNAs can cyclize (calculated delta G less than -11 kcal; 1 kcal = 4.184 kJ).

  6. 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

  7. Challenging the Roles of NSP3 and Untranslated Regions in Rotavirus mRNA Translation

    PubMed Central

    Gratia, Matthieu; Vende, Patrice; Charpilienne, Annie; Baron, Hilma Carolina; Laroche, Cécile; Sarot, Emeline; Pyronnet, Stéphane; Duarte, Mariela; Poncet, Didier

    2016-01-01

    Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex for rotavirus mRNAs. To further explore the effects of NSP3 and untranslated regions (UTRs) on rotavirus mRNAs translation, we used a quantitative in vivo assay with simultaneous cytoplasmic NSP3 expression (wild-type or deletion mutant) and electroporated rotavirus-like and standard synthetic mRNAs. This assay shows that the last four GACC nucleotides of viral mRNA are essential for efficient translation and that both the NSP3 eIF4G- and RNA-binding domains are required. We also show efficient translation of rotavirus-like mRNAs even with a 5’UTR as short as 5 nucleotides, while more than eleven nucleotides are required for the 3’UTR. Despite the weak requirement for a long 5’UTR, a good AUG environment remains a requirement for rotavirus mRNAs translation. PMID:26727111

  8. An element in the bovine papillomavirus late 3' untranslated region reduces polyadenylated cytoplasmic RNA levels.

    PubMed

    Furth, P A; Baker, C C

    1991-11-01

    Expression of the two bovine papillomavirus type 1 (BPV-1) late genes, L1 and L2, coding for the two capsid proteins, is limited to terminally differentiated keratinocytes in bovine fibropapillomas. This pattern of expression is determined both by the activity of the late promoter and by the inhibition of late region expression in less well differentiated cells. Inhibition of L1 and L2 mRNA production in nonpermissive cells must occur since the late region potentially could be transcribed from early region promoters. Nuclear runoff analysis of the late region has demonstrated that up to 95% of transcripts which are initiated in the early region in nonpermissive cells terminate within the late region upstream of the late polyadenylation site (C. C. Baker and J. Noe, J. Virol. 63:3529-3534, 1989). However, very few of the primary transcripts which include the late polyadenylation site are processed into mRNA. In this study, we have used expression vectors to characterize an inhibitory element active in nonpermissive cells which is located in the late 3' untranslated region (3'UTR). While the late polyadenylation site is functional in these cells, a 53-bp element in the late 3'UTR reduces levels of polyadenylated cytoplasmic RNA. This element inhibited chloramphenicol acetyltransferase (CAT) expression 6- to 10-fold when cloned in the sense orientation into the 3'UTR of a CAT expression vector. No block to expression was seen when the fragment was cloned immediately downstream of the poly(A) site, in an intron upstream of the CAT coding sequence, or in an antisense orientation in the 3'UTR. When the same fragment was deleted from a BPV-1 L1 expression vector, a sixfold increase in mRNA levels was seen. Actinomycin D chase experiments using BPV-1 L1 expression vectors indicated that the element does not destabilize cytoplasmic polyadenylated RNA. Therefore, the element must act before the mature mRNA reaches the cytoplasm. The data presented are consistent with effects

  9. Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements.

    PubMed

    Ozretić, Petar; Bisio, Alessandra; Musani, Vesna; Trnski, Diana; Sabol, Maja; Levanat, Sonja; Inga, Alberto

    2015-01-01

    PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway. PMID:25826662

  10. Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.

    PubMed

    Rubtsova, Maria P; Sizova, Daria V; Dmitriev, Sergei E; Ivanov, Dmitri S; Prassolov, Vladimir S; Shatsky, Ivan N

    2003-06-20

    A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

  11. Conserved nucleotide sequences in the open reading frame and 3' untranslated region of selenoprotein P mRNA.

    PubMed Central

    Hill, K E; Lloyd, R S; Burk, R F

    1993-01-01

    Rat liver selenoprotein P contains 10 selenocysteine residues in its primary structure (deduced). It is the only selenoprotein characterized to date that has more than one selenocysteine residue. Selenoprotein P cDNA has been cloned from human liver and heart cDNA libraries and sequenced. The open reading frames are identical and contain a signal peptide, indicating that the protein is secreted by both organs and is therefore not exclusively produced in the liver. Ten selenocysteine residues (deduced) are present. Comparison of the open reading frame of the human cDNA with the rat cDNA reveals a 69% identity of the nucleotide sequence and 72% identity of the deduced amino acid sequence. Two regions in the 3' untranslated portion have high conservation between human and rat. Each of these regions contains a predicted stable stem-loop structure similar to the single stem-loop structures reported in 3' untranslated regions of type I iodothyronine 5'-deiodinase and glutathione peroxidase. The stem-loop structure of type I iodothyronine 5'-deiodinase has been shown to be necessary for incorporation of the selenocysteine residue at the UGA codon. Because only two stem-loop structures are present in the 3' untranslated region of selenoprotein P mRNA, it can be concluded that a separate stem-loop structure is not required for each selenocysteine residue. Images PMID:8421687

  12. 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

  13. 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.

  14. 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.

  15. Role of 3’-untranslated region translational control in cancer development, diagnostics and treatment

    PubMed Central

    Vislovukh, Andrii; Vargas, Thaiz Rivera; Polesskaya, Anna; Groisman, Irina

    2014-01-01

    The messenger RNA 3’-untranslated region (3’UTR) plays an important role in regulation of gene expression on the posttranscriptional level. The 3’UTR controls gene expression via orchestrated interaction between the structural components of mRNAs (cis-element) and the specific trans-acting factors (RNA binding proteins and non-coding RNAs). The crosstalk of these factors is based on the binding sequences and/or direct protein-protein interaction, or just functional interaction. Much new evidence that has accumulated supports the idea that several RNA binding factors can bind to common mRNA targets: to the non-overlapping binding sites or to common sites in a competitive fashion. Various factors capable of binding to the same RNA can cooperate or be antagonistic in their actions. The outcome of the collective function of all factors bound to the same mRNA 3’UTR depends on many circumstances, such as their expression levels, affinity to the binding sites, and localization in the cell, which can be controlled by various physiological conditions. Moreover, the functional and/or physical interactions of the factors binding to 3’UTR can change the character of their actions. These interactions vary during the cell cycle and in response to changing physiological conditions. Abnormal functioning of the factors can lead to disease. In this review we will discuss how alterations of these factors or their interaction can affect cancer development and promote or enhance the malignant phenotype of cancer cells. Understanding these alterations and their impact on 3’UTR-directed posttranscriptional gene regulation will uncover promising new targets for therapeutic intervention and diagnostics. We will also discuss emerging new tools in cancer diagnostics and therapy based on 3’UTR binding factors and approaches to improve them. PMID:24600513

  16. The 3' untranslated region of picornavirus RNA: features required for efficient genome replication.

    PubMed Central

    Rohll, J B; Moon, D H; Evans, D J; Almond, J W

    1995-01-01

    The role of the 3' untranslated region (3'UTR) in the replication of enteroviruses has been studied with a series of mutants derived from either poliovirus type 3 (PV3) or a PV3 replicon containing the reporter gene chloramphenicol acetyltransferase. Replication was observed when the PV3 3'UTR was replaced with that of either coxsackie B4 virus, human rhinovirus 14 (HRV14), bovine enterovirus, or hepatitis A virus, despite the lack of sequence and secondary structure homology of the 3'UTRs of these viruses. The levels of replication observed for recombinants containing the 3'UTRs of hepatitis A virus and bovine enterovirus were lower than those for PV3 and the other recombinants. Extensive site-directed mutagenesis of the single stem-loop structure formed by the HRV14 3'UTR indicated the importance of (i) the loop sequence, (ii) the stability of the stem, and (iii) the location of the stem immediately upstream of the poly(A) tail. The role of a 4-bp motif at the base of the HRV14 stem, highly conserved among rhinoviruses, was examined by site-directed mutagenesis of individual base pairs. This analysis did not pinpoint a particular base pair as crucial for function. The requirement for immediate adjacent positioning of the open reading frame and the 3'UTR was examined by insertion of a 1.1-kb heterologous sequence. A replicon containing this insert replicated to about 30% of the level observed for the wild type. However, the corresponding virus consistently deleted most of the inserted fragment, suggesting that its presence was incompatible with a full replication cycle. PMID:7494295

  17. 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

  18. 3{prime} UTR sequence-specific mRNA-protein complexes and the post-transcriptional regulation of catalase

    SciTech Connect

    Reimer, D.L.; Ott, R.N.; Singh, S.M.

    1994-09-01

    Recently, sequences in the 3{prime} untranslated region (3{prime} UTR) of some genes have been recognized which may play an important role in the post-transcriptional regulation of gene expression. Mutations in this region of the gene are known to cause at least two diseases including myotonic dystrophy and a lysosomal accumulation disease. The mechanism is thought to involve mRNA-protein interactions that affect translation and/or mRNA stability. Reports of this nature are not common and the significance of the often large 3{prime} UTR on the regulation of gene expression remains speculative. Studies on the 3{prime} UTR mRNA-protein interactions in model eukaryotic genes therefore are critical to better understand the molecular mechanisms involved in post-transcriptional gene regulation. Mouse catalase, encoded by Cas-1, was used as a model to characterize the molecular mechanisms of post-transcriptional gene regulation. The 3{prime} UTR (752 bp) of Cas-1 contains three unusual, near repeats [(CA){sub 31}, (U){sub 15} and (TGTGC){sub 7}]. Gel mobility shift assays using {sup 32}P-labelled transcripts which contain these sequences and tissue homogenates from various sources identified mRNA-protein complexes specific to (CA){sub 31} and (U){sub 15} only. In all strains analyzed, a single protein of 69 kDa which was involved in the (CA){sub 31} complex, was observed in most tissues except lung and was localized to the polysomal fraction. Similarly, two proteins involved in the (U){sub 15} complex, 38 and 47 kDa, were observed in all tissues and strains studied. Only the 38 kDa protein was observed in the polysomal fraction. The results argue for a possible role for these 3{prime} UTR mRNA-binding protein complexes in the post-transcriptional regulation of this antioxidant enzyme.

  19. Targeting of c-myc and beta-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3' untranslated region.

    PubMed Central

    Hesketh, J; Campbell, G; Piechaczyk, M; Blanchard, J M

    1994-01-01

    The influence of the 3' untranslated region on mRNA localization was investigated by measuring the distribution of myc, beta-globin and hybrid myc-globin mRNAs between free, cytoskeletal-bound and membrane-bound polysomes in cells transfected with either control or chimeric gene constructs. c-myc sequences and beta-globin-coding sequences linked to the myc 3' untranslated region were present at greatest enrichment in cytoskeletal-bound polysomes. beta-Globin mRNA and myc-coding sequences linked to the beta-globin 3' untranslated region were recovered largely in the free polysomes. In situ hybridization confirmed that replacement of the c-myc 3' untranslated region by that of globin caused a relocalization of the mRNA. The results suggest that mRNA localization in differentiated eukaryotic cells depends on a mechanism that involves directional information in the 3' untranslated region of mRNAs. Images Figure 2 Figure 3 PMID:8129712

  20. 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.

  1. Recent Insights and Novel Bioinformatics Tools to Understand the Role of MicroRNAs Binding to 5′ Untranslated Region

    PubMed Central

    Da Sacco, Letizia; Masotti, Andrea

    2013-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through the binding of the 3′ untranslated region (3′UTR) of specific mRNAs. MiRNAs are post-transcriptional regulators and determine the repression of translation processes or the degradation of mRNA targets. Recently, another kind of miRNA-mediated regulation of translation (repression or activation) involving the binding of miRNA to the 5′UTR of target gene has been reported. The possible interactions and the mechanism of action have been reported in many works that we reviewed here. Moreover, we discussed also the available bioinformatics tools for predicting the miRNA binding sites in the 5′UTR and public databases collecting this information. PMID:23271365

  2. Similar interactions of the poliovirus and rhinovirus 3D polymerases with the 3' untranslated region of rhinovirus 14.

    PubMed

    Meredith, J M; Rohll, J B; Almond, J W; Evans, D J

    1999-12-01

    We showed previously that a human rhinovirus 14 (HRV14) 3' untranslated region (3' UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J. Virol 69:7835-7844, 1995). This enabled the HRV14 single 3' UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3' UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3' UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3' UTR of rhinovirus with both poliovirus and rhinovirus polymerases.

  3. The 5'-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation.

    PubMed Central

    Rohll, J B; Percy, N; Ley, R; Evans, D J; Almond, J W; Barclay, W S

    1994-01-01

    The role of the 5'-untranslated region (5'UTR) in the replication of enteroviruses has been studied by using a series of poliovirus type 3 (PV3) replicons containing the chloramphenicol acetyltransferase reporter gene in which the 5'UTR was replaced by the 5'UTR of either coxsackievirus B4 or human rhinovirus 14 or composite 5'UTRs derived from sequences of PV3, human rhinovirus 14, coxsackievirus B4, or encephalomyocarditis virus. The results indicate that efficient replication of an enterovirus genome requires a compatible interaction between the 5'-terminal cloverleaf structure and the coding and/or 3'-noncoding regions of the genome. A crucial determinant of this interaction is the stem-loop formed by nucleotides 46 to 81 (stem-loop d). The independence of the cloverleaf structure formed by the 5'-terminal 88 nucleotides and the ribosome landing pad or internal ribosome entry site (IRES) was investigated by constructing a 5'UTR composed of the PV3 cloverleaf and the IRES from encephalomyocarditis virus. Chloramphenicol acetyltransferase gene-containing replicons and viruses containing this recombinant 5'UTR showed levels of replication similar to those of the corresponding genomes containing the complete PV3 5'UTR, indicating that the cloverleaf and the IRES may be regarded as functionally independent and nonoverlapping elements. Images PMID:8207812

  4. Cloning and characterization of 5'-untranslated region of porcine beta casein gene (CSN2).

    PubMed

    Lee, Poongyeon; Chung, Hee Kyoung; Lee, Hyun-Gi; Lee, Hwi-Cheul; Woo, Jae-Seok; Lee, Seunghoon; Jo, Su-Jin; Chang, Won-Kyong; Lee, Hoon-Taek; Kwon, Moosik; Park, Jin-Ki

    2008-10-01

    beta-Casein (CSN2) is a major milk protein in most mammals. The CSN2 gene is generally induced by lactogenic hormones bound to its promoter. The expression of this gene can be enhanced by signal transducers and activators of transcription (STAT) and glucocorticoid receptor (GR). Here, we analyzed the promoter and intron 1 regions of the porcine CSN2 gene. The porcine CSN2 promoter and intron 1 regions (-3098bp to +2446bp) were cloned into the pGL3-Basic vector containing the luciferase reporter gene (pCSN2-PEI). Lactogenic signals induced the transcription of porcine CSN2. By using AG490, a Janus kinase (JAK) inhibitor, we demonstrated that STAT5 positively regulates the transcription of porcine CSN2. Further, seven STAT mutants were generated by site-directed mutagenesis. By performing electrophoretic mobility shift assays (EMSAs), we located a critical element for pCSN2-PEI transcription bound to STAT5 in the -102bp to -84bp region. The construct containing only the promoter region (pCSN2-P), however, did not exert any promotive effects on transcription in two cell types-a mouse mammary epithelial cell line (HC11) and porcine mammary gland epithelial cells (PMECs). Thus, the construct containing intron 1 of porcine CSN2 exerts an elevating effect on transcription. We suggest that the transcription of porcine CSN2 is regulated by lactogenic signals via the STAT5 site (-102bp to -84bp) and intron 1.

  5. Sole and stable RNA duplexes of G-rich sequences located in the 5'-untranslated region of protooncogenes.

    PubMed

    Saxena, Sarika; Miyoshi, Daisuke; Sugimoto, Naoki

    2010-08-24

    Guanine- (G-) rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. It is widely held that the formation of a G-quadruplex in RNA is more feasible than in DNA because of the lack of a complementary strand in mRNA. Here, we analyzed sequences of 5'-untranslated regions of protooncogenes and surprisingly found that these regions showed an enrichment of not only guanine (G) but also cytosine (C) nucleotides. Since neighboring cytosine- (C-) rich regions can affect the formation and stability of a G-quadruplex structure, we further investigated the properties of DNA and RNA structures of G-rich and GC-rich regions. We selected typical GC-rich RNA sequences from protooncogenes and corresponding DNA sequences and investigated their structures. It was found that the GC-rich RNA sequences formed stable A-form duplexes as their major structure independent of the surrounding conditions, including the presence of different cations (Na(+), K(+), or Li(+)) or molecular crowding with 40 wt % poly(ethylene glycol) with an average molecular mass of 200 Da although there are a few exceptions in which only a combination of K(+) and molecular crowding induced a G-quadruplex structure of an extremely G-rich RNA sequence. In contrast, structural polymorphisms involving duplexes, G-quadruplexes, and i-motifs were observed for GC-rich DNA sequences depending on the surrounding factors. These results demonstrate the considerable structural and functional differences in GC-rich sequences of the genome (DNA) and transcriptosome (mRNA) with respect to the nucleic acid backbone. Moreover, it was suggested that structural study for a G-rich RNA sequence should be carried out under cell-mimicking condition where K(+) and crowding cosolutes exist.

  6. HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.

    PubMed

    Stake, Matthew; Singh, Deepali; Singh, Gatikrushna; Marcela Hernandez, J; Kaddis Maldonado, Rebecca; Parent, Leslie J; Boris-Lawrie, Kathleen

    2015-12-01

    Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.

  7. EFFECTS OF 5’ UNTRANSLATED REGION DIVERSITY ON THE POSTTRANSCRIPTIONAL REGULATION OF THE HUMAN REDUCED FOLATE CARRIER

    PubMed Central

    Payton, Scott G.; Haska, Christina L.; Flatley, Robin M.; Ge, Yubin; Matherly, Larry H.

    2007-01-01

    The human RFC (hRFC) gene is regulated by five major 5’ non-coding exons, characterized by alternate transcription start sites and splice forms. The result is up to 14 hRFC transcripts for which different 5’ untranslated regions (UTRs) are fused to a common coding sequence. By in vitro translation assays with hRFC constructs corresponding to the major transcript forms, most of the forms were translated poorly. Upon expression of the 5’UTR-hRFC constructs in hRFC-null HeLa cells, a range of steady state hRFC proteins and transcripts were detected that reflected relative transcript stabilities and, to a lesser extent, translation efficiencies. Transcripts including 5’ UTRs derived from non-coding exon A encoded a modified hRFC protein translated from an upstream initiation site. When this modified hRFC protein was expressed in hRFC-null K562 cells, there were only minor differences in surface targeting, stability, or transport function from wild type hRFC. Our results demonstrate an important role for posttranscriptional determinants of cellular hRFC levels and activity. PMID:17306382

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

    PubMed

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

    2015-09-08

    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.

  9. 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

  10. Effects of 3′ Untranslated Region Mutations on Plus-Strand Priming during Moloney Murine Leukemia Virus Replication

    PubMed Central

    Robson, Nicole D.; Telesnitsky, Alice

    1999-01-01

    A conserved purine-rich motif located near the 3′ end of retroviral genomes is involved in the initiation of plus-strand DNA synthesis. We mutated sequences both within and flanking the Moloney murine leukemia virus polypurine tract (PPT) and determined the effects of these alterations on viral DNA synthesis and replication. Our results demonstrated that both changes in highly conserved PPT positions and a mutation that left only the cleavage-proximal half of the PPT intact led to delayed replication and reduced the colony-forming titer of replication defective retroviral vectors. A mutation that altered the cleavage proximal half of the PPT and certain 3′ untranslated region mutations upstream of the PPT were incompatible with or severely impaired viral replication. To distinguish defects in plus-strand priming from other replication defects and to assess the relative use of mutant and wild-type PPTs, we examined plus-strand priming from an ectopic, secondary PPT inserted in U3. The results demonstrated that the analyzed mutations within the PPT primarily affected plus-strand priming whereas mutations upstream of the PPT appeared to affect both plus-strand priming and other stages of viral replication. PMID:9882295

  11. Efficient inhibition of porcine reproductive and respiratory syndrome virus replication by artificial microRNAs targeting the untranslated regions.

    PubMed

    Xia, Bing; Song, Hongqin; Chen, Yang; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2013-01-01

    A robust artificial microRNA (amiRNA) strategy against porcine reproductive and respiratory syndrome virus (PRRSV) was developed by targeting the untranslated regions (UTRs). Six candidate amiRNAs targeting the 5' or 3' UTR were used for vector construction, and four effective amiRNAs were selected for further study using a vector transfection/virus infection assay. In cell cultures stably transfected with the four amiRNA vectors, expression of the sequence-specific amiRNAs was confirmed using poly(A)-tailed RT-PCR. After infection with three different PRRSV strains, the viral RNA genome and/or transcript were inhibited by ~90 % (semi-quantitative RT-PCR), and the viral titers were decreased by more than six log CCID(50) (viral titration assay) before day 3 postinfection. The potent anti-PRRSV effects lasted for at least 5 days. Sequence analysis showed that the amiRNA antiviral activities were not compromised by the presence of one or two mismatches in their binding targets. This work constitutes a step towards developing a more effective RNAi strategy against PRRSV.

  12. 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.

  13. How does a scanning ribosomal particle move along the 5'-untranslated region of eukaryotic mRNA? Brownian Ratchet model.

    PubMed

    Spirin, Alexander S

    2009-11-17

    A model of the ATP-dependent unidirectional movement of the 43S ribosomal initiation complex (=40S ribosomal subunit + eIF1 + eIF1A + eIF2.GTP.Met-tRNA(i) + eIF3) during scanning of the 5'-untranslated region of eukaryotic mRNA is proposed. The model is based on the principles of molecular Brownian ratchet machines and explains several enigmatic data concerning the scanning complex. In this model, the one-dimensional diffusion of the ribosomal initiation complex along the mRNA chain is rectified into the net-unidirectional 5'-to-3' movement by the Feynman ratchet-and-pawl mechanism. The proposed mechanism is organized by the heterotrimeric protein eIF4F (=eIF4A + eIF4E + eIF4G), attached to the scanning ribosomal particle via eIF3, and the RNA-binding protein eIF4B that is postulated to play the role of the pawl. The energy for the useful work of the ratchet-and-pawl mechanism is supplied from ATP hydrolysis induced by the eIF4A subunit: ATP binding and its hydrolysis alternately change the affinities of eIF4A for eIF4B and for mRNA, resulting in the restriction of backward diffusional sliding of the 43S ribosomal complex along the mRNA chain, while stochastic movements ahead are allowed.

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

    PubMed

    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.

  15. 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

  16. 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

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

    PubMed

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

    2013-03-01

    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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25002673

  20. HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.

    PubMed

    Stake, Matthew; Singh, Deepali; Singh, Gatikrushna; Marcela Hernandez, J; Kaddis Maldonado, Rebecca; Parent, Leslie J; Boris-Lawrie, Kathleen

    2015-12-01

    Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages. PMID:26584240

  1. 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

  2. Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation.

    PubMed

    Verrier, S B; Jean-Jean, O

    2000-04-01

    In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.

  3. 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

  4. A regulatory role for the 5' and 3' untranslated regions in differential expression of hsp83 in Leishmania.

    PubMed Central

    Aly, R; Argaman, M; Halman, S; Shapira, M

    1994-01-01

    Exposure of Leishmania promastigotes to temperatures typical of mammals result in a stress response, which is accompanied by an increase in the steady state level of heat shock transcripts and their translation. Accumulation of the heat shock protein (hsp83) mRNA occurs due to differential decay rates at the altered temperatures, while transcription is unaffected. A similar pattern of post-transcriptional regulation was observed for a transfected chloramphenicol acetyltransferase (CAT) gene, which was flanked at both ends by intergenic regions (IR) of hsp83. Shortening the 5' untranslated region (UTR) by 100 nts produced an active CAT enzyme, but abolished the temperature-dependent regulation of the CAT-hsp83 mRNA turn-over. The 3' UTR is also involved in the temperature-dependent degradation of hsp83 mRNA, since exchange of the hsp83 3' UTR with a parallel fragment from a non-heat shock gene abolished the differential turn-over of CAT mRNA. Thus, the regulated decay of hsp83 mRNA is controlled by sequence or conformational elements present in both upstream and downstream UTRs. Like the endogenous hsp83, translation of CAT mRNA which contained hsp83 UTRs was higher at 35 degrees C. This was observed only with transcripts in which stability increased at elevated temperatures. Modifications which abolished the temperature dependence of CAT mRNA decay, eliminated its elevated translation at the higher temperatures. The correlation suggests a mechanistic link between the translational machinery and mRNA stability. Images PMID:8065903

  5. Enhancer activity of light-responsive regulatory elements in the untranslated leader regions of cyanobacterial psbA genes.

    PubMed Central

    Li, R; Golden, S S

    1993-01-01

    Three psbA genes encoding the D1 protein of the photosystem II reaction center are differentially expressed under different light intensities in the cyanobacterium Synechococcus sp. strain PCC 7942. Two of the three psbA genes, psbAII and psbAIII, are induced rapidly when light intensity is increased from 125 x 10(-6) mol.m-2.s-1 to 750 x 10(-6) mol.m-2.s-1. A recombinational cloning vector that carries a transcriptional lacZ reporter gene was used to characterize the controlling elements responsible for light induction. At least three distinct cis elements are present in the regulatory regions of pbsAII and psbAIII: basal promoters, comparable to Escherichia coli sigma 70 promoters in position and sequence, confer constitutive expression of the genes under both low and high light intensities; negative elements upstream of the promoters down-regulate the expression of the corresponding gene; and sequences downstream of the promoters that correspond to the untranslated leader regions of the mRNAs (+1 to +41 in psbAII and +1 to +39 in psbAIII) are responsible for increased expression under high light. When these light-responsive elements were combined with an E. coli promoter (conII) in different positions and orientations, the expression of the lacZ gene was induced 4- to 11-fold. The induction of gene expression under high light by these enhancers was position independent but orientation dependent. When the elements were combined with the conII promoter in the correct orientation, they also conferred a small but reproducible level of light-responsive expression on this E. coli promoter. Images Fig. 1 Fig. 3 Fig. 4 PMID:8265608

  6. The 5′ Untranslated Region of a Novel Infectious Molecular Clone of the Dicistrovirus Cricket Paralysis Virus Modulates Infection

    PubMed Central

    Kerr, Craig H.; Wang, Qing S.; Keatings, Kathleen; Khong, Anthony; Allan, Douglas; Yip, Calvin K.

    2015-01-01

    ABSTRACT 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. IMPORTANCE Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host

  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. 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.

  9. 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

  10. 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

  11. 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.

  12. Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

    PubMed Central

    Hann, L E; Webb, A C; Cai, J M; Gehrke, L

    1997-01-01

    We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis. PMID:9121448

  13. 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. PMID:26839368

  14. Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5′-Untranslated-Region Mutants

    PubMed Central

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

    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. PMID:24173235

  15. Multiple Regulatory Mechanisms Act on the 5′ Untranslated Region of the S-Layer Gene from Thermus thermophilus HB8

    PubMed Central

    Castán, Pablo; de Pedro, Miguel A.; Risco, Cristina; Vallés, Cristina; Fernández, Luis A.; Schwarz, Heinz; Berenguer, José

    2001-01-01

    The role of the 5′ untranslated region (5′UTR) of the S-layer gene from Thermus thermophilus was analyzed through the isolation of Δ5′UTR mutants. In these mutants the half-life of splA mRNA was strongly reduced and slpA transcription was no longer subjected to growth phase-dependent repression. Overproduction and detachment of the external envelopes of the mutants were observed in stationary phase. PMID:11157968

  16. Fragile X Mental Retardation Protein Interactions with a G quadruplex structure in the 3′-Untranslated Region of NR2B mRNA

    PubMed Central

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

    2015-01-01

    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. PMID:26412477

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

    PubMed Central

    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 18bp 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 +247bp and +266bp 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 18bp 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 18bp segment. Hence, the data presented here demonstrate that the 18bp indel in the porcine SOX9 5′-UTR is of functional importance and

  18. 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

  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. PMID:27233801

  20. 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

  1. Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein.

    PubMed Central

    Thekkumkara, Thomas J; Linas, Stuart L

    2003-01-01

    The mRNA 3'-untranslated region (3'-UTR) of many genes has been identified as an important regulator of the mRNA transcript itself as well as the translated product. Previously, we demonstrated that Chinese-hamster ovary-K1 cells stably expressing angiotensin receptor subtypes (AT(1A)) with and without 3'-UTR differed in AT(1A) mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT(1A) 3'-UTR probe identified a major mRNA-binding protein complex of 55 kDa in Chinese-hamster ovary-K1 cells. In the present study, we have determined the functional significance of the native AT(1A) receptor 3'-UTR in rat liver epithelial (WB) cell lines by co-expressing the AT(1A) 3'-UTR sequence 'decoy' to compete with the native receptor 3'-UTR for its mRNA-binding proteins. PCR analysis using specific primers for the AT(1A) receptor and [(125)I]angiotensin II (AngII)-binding studies demonstrated the expression of the native AT(1A) receptors in WB (B(max)=2.7 pmol/mg of protein, K(d)=0.56 nM). Northern-blot analysis showed a significant increase in native receptor mRNA expression in 3'-UTR decoy-expressing cells, confirming the role of 3'-UTR in mRNA destabilization. Compared with vehicle control, AngII induced DNA and protein synthesis in wild-type WB as measured by [(3)H]thymidine and [(3)H]leucine incorporation respectively. Activation of [(3)H]thymidine and [(3)H]leucine correlated with a significant increase in cell number (cellular hyperplasia). In these cells, AngII stimulated GTPase activity by AT(1) receptor coupling with G-protein alpha i. We also delineated that functional coupling of AT(1A) receptor with G-protein alpha i is an essential mechanism for AngII-mediated cellular hyperplasia in WB by specifically blocking G-protein alpha i activation. In contrast with wild-type cells, stable expression of the 3'-UTR 'decoy' produced AngII-stimulated protein synthesis and cellular

  2. 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.

  3. The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

    PubMed

    Gallie, D R; Young, T E

    1994-11-01

    Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

  4. Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha.

    PubMed Central

    Alonso, J; Sánchez de Miguel, L; Montón, M; Casado, S; López-Farré, A

    1997-01-01

    Changes in endothelial nitric oxide synthase (eNOS) expression may be involved in the endothelium-dependent vasorelaxation dysfunction associated with several vascular diseases. In the present work, we demonstrate that eNOS mRNA contains a previously undescribed cis element in the 3' untranslated region (3' UTR). A U+C-rich segment in the 3' UTR is critical in complex formation with bovine aortic endothelial cell cytosolic proteins. Tumor necrosis factor alpha (TNF-alpha), which destabilizes eNOS mRNA, increased the binding activity of the cytosolic proteins in a time-dependent manner. These data suggest that endothelial cytosolic proteins bind to the 3' UTR of eNOS mRNA. These proteins may play a role in TNF-alpha-induced eNOS mRNA destabilization. PMID:9315630

  5. 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

  6. 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-.

  7. Comparative analysis reveals no consistent association between the secondary structure of the 3'-untranslated region of dengue viruses and disease syndrome.

    PubMed

    Zhou, Yang; Mammen, Mammen P; Klungthong, Chonticha; Chinnawirotpisan, Piyawan; Vaughn, David W; Nimmannitya, Suchitra; Kalayanarooj, Siripen; Holmes, Edward C; Zhang, Chunlin

    2006-09-01

    A comparative analysis was performed of the 3'-untranslated region (UTR) of Dengue virus (DENV) sampled from Bangkok, Thailand, over a 30 year period and representing all four serotypes. Considerable genetic variation was observed both within and among serotypes. Notably, a full-length version of the critical 3'-long stable hairpin structure was absent from some isolates, suggesting the occurrence of complex structural interactions within the 3'-UTR, including the influence of upstream mutations. The Thai sequences were then combined with 61 globally sampled isolates of DENV taken from patients with either dengue fever or severe dengue disease. No consistent association was found between 3'-UTR secondary structure and the clinical outcome of DENV infection, although some evidence for a trend in this direction was observed in DENV-2. It was concluded that the 3'-UTR is not the sole determinant of DENV virulence in nature, although variation in secondary structure may greatly influence viral fitness.

  8. Posttranscriptional control of photosynthetic mRNA decay under stress conditions requires 3' and 5' untranslated regions and correlates with differential polysome association in rice.

    PubMed

    Park, Su-Hyun; Chung, Pil Joong; Juntawong, Piyada; Bailey-Serres, Julia; Kim, Youn Shic; Jung, Harin; Bang, Seung Woon; Kim, Yeon-Ki; Do Choi, Yang; Kim, Ju-Kon

    2012-07-01

    Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3' and 5' untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3' and 5' untranslated regions and correlates with differential polysome

  9. Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA.

    PubMed

    Czyzyk-Krzeska, M F; Dominski, Z; Kole, R; Millhorn, D E

    1994-04-01

    Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed. PMID:7908289

  10. Ca2+ ionophore A23187-dependent stabilization of granulocyte-macrophage colony-stimulating factor messenger RNA in murine thymoma EL-4 cells is mediated through two distinct regions in the 3'-untranslated region.

    PubMed

    Iwai, Y; Akahane, K; Pluznik, D H; Cohen, R B

    1993-05-15

    We analyze the role of the Ca2+ ionophore A23187 in the induction of GM-CSF mRNA expression in EL-4 thymoma cells. Northern analysis shows that A23187 increases the half-life of GM-CSF mRNA. To identify potential Ca2+ response elements in the GM-CSF mRNA, we produced stable transfectants containing pRSV-CAT (EL-4cat) or hybrid constructs in which most of the GM-CSF 3'-untranslated region (EL-4gm) or the adenosine-uridine boxes alone (EL-4au) were placed in a downstream position from the CAT coding region. A23187 induces a 4.4-fold increase in CAT activity in EL-4cat cells and a 210-fold and 48-fold increase in CAT activity in EL-4gm and EL-4au cells, respectively. Actinomycin D chase experiments in transfected cells demonstrate that A23187 increases the half-life of CAT mRNA from 15 min to 3 h in EL-4au cells and more than 3 h in EL-4gm cells, suggesting that the effect of Ca2+ is mediated predominantly by the adenosine-uridine boxes with a smaller contribution from upstream regions. To map these upstream regions, we transfected cells with constructs containing mutations of the 3'-untranslated region. With two of these mutations, corresponding to a region located about 160 bases upstream of the adenosine-uridine boxes, CAT activity was induced only 50-fold compared to 200-fold in EL-4gm cells. These data indicate that two regions within the GM-CSF 3'-untranslated region interact to modulate Ca2+ effects on GM-CSF mRNA half-life. PMID:8482841

  11. 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

  12. 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.

  13. Molecular Archaeology of Flaviviridae Untranslated Regions: Duplicated RNA Structures in the Replication Enhancer of Flaviviruses and Pestiviruses Emerged via Convergent Evolution

    PubMed Central

    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. PMID:24647143

  14. HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

    PubMed Central

    Yu, Rong; Yang, Darong; Lei, Shaohua; Wang, Xiaohong; Meng, Xianghe; Xue, Binbin

    2015-01-01

    ABSTRACT High-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome. IMPORTANCE Hepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is

  15. Determination of the secondary structure of and cellular protein binding to the 3'-untranslated region of the hepatitis C virus RNA genome.

    PubMed Central

    Ito, T; Lai, M M

    1997-01-01

    Hepatitis C virus (HCV) contains a positive-stranded RNA genome of approximately 9.5 kb. Despite the overall sequence diversity among individual HCV isolates, the 3'-end 98 nucleotides (nt) of the HCV RNA, which constitute part of the 3'-untranslated region (3'-UTR), are highly conserved. This conserved region may contain the cis-acting signals for RNA replication involving possibly both viral and cellular proteins. We carried out RNase digestion studies, which revealed that this 98-nt region contains three stem-loops but may also assume alternative structures. We further performed UV cross-linking experiments to detect cellular proteins that bound to this region. A 58-kDa cellular protein (p58) was detected. Its binding site was mapped to the stem-loops 2 and 3, which are the most conserved region of the 3'-UTR. Site-directed mutagenesis studies revealed that both stem structures and specific nucleotide sequence within the two loops are important for p58 binding. Mutations that disrupted stem structures abolished protein binding, while the compensatory mutations restored its binding. This region also contains partial sequence similarity to the reported consensus binding sequence for polypyrimidine tract-binding protein (PTB) (a 57-kDa protein). The UV-cross-linked protein could be immunoprecipitated with the anti-PTB antibody, and the recombinant PTB bound to the HCV 3'-UTR with the same binding specificity as p58, establishing that this protein is PTB. However, the reported PTB-binding sequence was not sufficient, but rather the entire stem-loops 2 and 3 were required, for PTB binding; thus, its binding specificity is significantly different from the reported PTB-binding sequence requirement. This protein was detected in both the nuclei and cytoplasm of most mammalian cell lines tested and human primary hepatocytes. PTB may participate in the regulation of HCV RNA synthesis or translation. PMID:9343228

  16. 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

  17. 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

  18. An element in the 3' untranslated region of human LINE-1 retrotransposon mRNA binds NXF1(TAP) and can function as a nuclear export element.

    PubMed Central

    Lindtner, Susan; Felber, Barbara K; Kjems, Jørgen

    2002-01-01

    Export of unspliced mRNA to the cytoplasm is required for the replication of all retroviruses. In simian type D retroviruses, the RNA export is mediated by the constitutive transport element (CTE) that binds the cellular nuclear export factor 1, NXF1(TAP). To search for potential cellular RNA substrates for NXF1, we have set up an in vitro selection procedure, using an RNA library expressed from total human genomic DNA. A sequence that was isolated most frequently as independent clones exhibits extensive homology to the 3' untranslated region of expressed LINE1 (L1) retrotransposons. This region, termed L1-NXF1 binding element (L1-NBE) bears no structural resemblance to the viral CTE, but binds NXF1 as strongly as CTE, based on gel mobility shift competition assays. A deletion analysis of the NXF1 protein reveals that CTE and L1-NBE have different, but overlapping, binding domains on NXF1. Placed in an intron, L1-NBE is capable of mediating nuclear export of lariat RNA species in Xenopus laevis oocytes and of an unspliced HIV-1 derived RNA in human 293 cells, suggesting that it may function as a nuclear export element for the intronless L1 mRNA. PMID:12003494

  19. Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3' untranslated region of alfalfa mosaic virus RNA 4.

    PubMed Central

    Houser-Scott, F; Baer, M L; Liem, K F; Cai, J M; Gehrke, L

    1994-01-01

    The specific binding of alfalfa mosaic virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus coat protein and ilavirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements. Images PMID:8139004

  20. 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. PMID:27291656

  1. Vimentin interacts with the 5′-untranslated region of mouse mu opioid receptor (MOR) and is required for post-transcriptional regulation

    PubMed Central

    Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

    2013-01-01

    The opioid receptors are among the most highly studied members of the superfamily of G-protein coupled receptors. Morphine and endogenous mu opioid peptides exert their pharmacological actions mainly through the mu opioid receptor (MOR). Expression of opioid receptor proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5′-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. Here, we demonstrate for the first time the role of vimentin as a post-transcriptional repressor in MOR gene regulation. To identify potential regulators of the mouse MOR gene, we performed affinity column chromatography using 5′-UTR-specific RNA oligonucleotides using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified an intermediate filament protein, vimentin, which bound specifically to the region between -175 and -150 (175–150) of the MOR 5′-UTR. Binding was confirmed by western blot analysis and RNA supershift assay. Furthermore, a cotransfection study demonstrated that the presence of vimentin resulted in reduced expression of the mouse MOR. Our data suggest that vimentin functions as a repressor of MOR translation, dependent on 175–150 of the MOR 5′-UTR. PMID:23353576

  2. 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.

  3. Ultra-deep sequencing analysis of the hepatitis A virus 5'-untranslated region among cases of the same outbreak from a single source.

    PubMed

    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

  4. HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

    PubMed Central

    Shwetha, Shivaprasad; Kumar, Anuj; Mullick, Ranajoy; Vasudevan, Deeptha; Mukherjee, Nilanjan

    2015-01-01

    ABSTRACT HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3′ untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3′ UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3′ UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3′ UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3′ untranslated region (UTR) of HCV RNA. At the 3′ UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. 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.

  7. 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

  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. Genomewide mapping and screening of Kaposi's sarcoma-associated herpesvirus (KSHV) 3' untranslated regions identify bicistronic and polycistronic viral transcripts as frequent targets of KSHV microRNAs.

    PubMed

    Bai, Zhiqiang; Huang, Yufei; Li, Wan; Zhu, Ying; Jung, Jae U; Lu, Chun; Gao, Shou-Jiang

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes over 90 genes and 25 microRNAs (miRNAs). The KSHV life cycle is tightly regulated to ensure persistent infection in the host. In particular, miRNAs, which primarily exert their effects by binding to the 3' untranslated regions (3'UTRs) of target transcripts, have recently emerged as key regulators of KSHV life cycle. Although studies with RNA cross-linking immunoprecipitation approach have identified numerous targets of KSHV miRNAs, few of these targets are of viral origin because most KSHV 3'UTRs have not been characterized. Thus, the extents of viral genes targeted by KSHV miRNAs remain elusive. Here, we report the mapping of the 3'UTRs of 74 KSHV genes and the effects of KSHV miRNAs on the control of these 3'UTR-mediated gene expressions. This analysis reveals new bicistronic and polycistronic transcripts of KSHV genes. Due to the 5'-distal open reading frames (ORFs), KSHV bicistronic or polycistronic transcripts have significantly longer 3'UTRs than do KSHV monocistronic transcripts. Furthermore, screening of the 3'UTR reporters has identified 28 potential new targets of KSHV miRNAs, of which 11 (39%) are bicistronic or polycistronic transcripts. Reporter mutagenesis demonstrates that miR-K3 specifically targets ORF31-33 transcripts at the lytic locus via two binding sites in the ORF33 coding region, whereas miR-K10a-3p and miR-K10b-3p and their variants target ORF71-73 transcripts at the latent locus through distinct binding sites in both 5'-distal ORFs and intergenic regions. Our results indicate that KSHV miRNAs frequently target the 5'-distal coding regions of bicistronic or polycistronic transcripts and highlight the unique features of KSHV miRNAs in regulating gene expression and life cycle. PMID:24155407

  10. Evidence implicating the 5' untranslated region of Listeria monocytogenes actA in the regulation of bacterial actin-based motility.

    PubMed

    Wong, Kendy K Y; Bouwer, H G Archie; Freitag, Nancy E

    2004-02-01

    The ActA protein of Listeria monocytogenes is a major virulence factor, essential for the recruitment and polymerization of host actin filaments that lead to intracellular motility and cell-to-cell spread of bacteria within the infected host. The expression of actA is tightly regulated and is strongly induced only when L. monocytogenes is within the host cytosol. Intracellular induction of actA expression is mediated through a single promoter element that directs the expression of a messenger RNA with a long (150 bp) 5' untranslated region (UTR). Deletion of the actA+3 to +130 upstream region was found to result in bacterial mutants that were no longer capable of intracellular actin recruitment or cell-to-cell spread, thus indicating that this region is important for actA expression. L. monocytogenes strains that contained smaller deletions (21-23 bp) within the actA upstream region demonstrated a range of actA expression levels that coincided with the amount of bacterial cell-to-cell spread observed within infected monolayers. A correlation appeared to exist between levels of actA expression and the ability of L. monocytogenes to transition from uniform actin accumulation surrounding individual bacteria (actin clouds) to directional assembly and the formation of actin tails. Bacterial mutants containing deletions that most significantly altered the predicted secondary structure of the actA mRNA 5' UTR had the largest reductions in actA expression. These results suggest that the actA 5' UTR is required for maximal ActA synthesis and that a threshold level of ActA synthesis must be achieved to promote the transition from bacteria-associated actin clouds to directional actin assembly and movement.

  11. 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.

  12. A three-nucleotide insertion in the H stem-loop of the 5' untranslated region of Theiler's virus attenuates neurovirulence.

    PubMed Central

    Bandyopadhyay, P K; Pritchard, A; Jensen, K; Lipton, H L

    1993-01-01

    The highly structured 5' untranslated region (5' UTR) of Theiler's murine encephalomyelitis virus is involved in cap-independent translation of the viral RNA. Previously, we reported that the bicistronic mRNA chloramphenicol acetyltransferase-5' UTR-luciferase (Luc) efficiently expressed Luc both in a rabbit reticulocyte lysate and when transfected into BHK-21 cells. Insertion of 3 nucleotides at position 665 in the 5' UTR of this bicistronic mRNA resulted in greatly reduced Luc expression in BHK-21 cells but had little effect on expression of Luc in rabbit reticulocyte lysate. This mutation was also introduced into a virulent Theiler's murine encephalomyelitis virus chimera, Chi-VL. The kinetics of viral RNA and protein synthesis and virus production in BHK-21 cells were slower for the mutant chimera [Chi-VL(IN668)] than for Chi-VL; however, the final virus yields were comparable. Intracerebral inoculation of mice with the chimeras revealed that Chi-VL(IN668) was completely attenuated in neurovirulence. The reduced neurovirulence of Chi-VL(IN668) may be ascribed to its reduced growth in the central nervous system, most likely due to an impaired ability to synthesize viral proteins. Images PMID:7684472

  13. A three-nucleotide insertion in the H stem-loop of the 5' untranslated region of Theiler's virus attenuates neurovirulence.

    PubMed

    Bandyopadhyay, P K; Pritchard, A; Jensen, K; Lipton, H L

    1993-06-01

    The highly structured 5' untranslated region (5' UTR) of Theiler's murine encephalomyelitis virus is involved in cap-independent translation of the viral RNA. Previously, we reported that the bicistronic mRNA chloramphenicol acetyltransferase-5' UTR-luciferase (Luc) efficiently expressed Luc both in a rabbit reticulocyte lysate and when transfected into BHK-21 cells. Insertion of 3 nucleotides at position 665 in the 5' UTR of this bicistronic mRNA resulted in greatly reduced Luc expression in BHK-21 cells but had little effect on expression of Luc in rabbit reticulocyte lysate. This mutation was also introduced into a virulent Theiler's murine encephalomyelitis virus chimera, Chi-VL. The kinetics of viral RNA and protein synthesis and virus production in BHK-21 cells were slower for the mutant chimera [Chi-VL(IN668)] than for Chi-VL; however, the final virus yields were comparable. Intracerebral inoculation of mice with the chimeras revealed that Chi-VL(IN668) was completely attenuated in neurovirulence. The reduced neurovirulence of Chi-VL(IN668) may be ascribed to its reduced growth in the central nervous system, most likely due to an impaired ability to synthesize viral proteins.

  14. 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

  15. 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.

  16. 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.

  17. CFTR mRNA expression is regulated by an upstream open reading frame and RNA secondary structure in its 5' untranslated region.

    PubMed

    Lukowski, Samuel W; Rothnagel, Joseph A; Trezise, Ann E O

    2015-02-15

    Post-transcriptional regulation of gene expression through 5' untranslated region (5'UTR)-encoded cis-acting elements is an important mechanism for the control of protein expression levels. Through controlling specific aspects of translation initiation, expression can be tightly regulated while remaining responsive to cellular requirements. With respect to cystic fibrosis (CF), the overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) protein trafficking mutants, such as delta-F508, is of great biological and clinical interest. By understanding the post-transcriptional mechanisms that regulate CFTR expression, new procedures can be developed to enhance CFTR expression in homozygous delta-F508 CF patients. We have identified the key elements of a complex negative regulatory mechanism that is encoded within the human CFTR 5'UTR and show how these elements act in combination to restrict CFTR gene expression to a consistently low level in a transcript-specific manner. This study shows, for the first time, that endogenous human CFTR expression is post-transcriptionally regulated through a 5'UTR-mediated mechanism. We show that the very low levels of endogenous CFTR expression, compared with other low expression genes, are maintained through the co-operative inhibitory effects of an upstream open reading frame and a thermodynamically stable RNA secondary structure. PMID:25274779

  18. 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

  19. Variant in the 5' untranslated region of insulin-like growth factor 1 receptor is associated with susceptibility to mastitis in cattle.

    PubMed

    Sugimoto, Mayumi; Sugimoto, Yoshikazu

    2012-09-01

    Mastitis is a common infectious disease of the mammary gland and generates large losses in the dairy industry. By means of positional cloning and functional analysis techniques, we here show that insulin-like growth factor 1 receptor (IGF1R) can possibly mediate susceptibility to mastitis through autophagy. Scanning the whole genome of cows (Bos taurus) that were susceptible or resistant to mastitis in the half-sib families revealed that susceptible cows had a relatively long stretch of cytosine residues (C stretch) in the 5' untranslated region of IGF1R. The forebrain embryonic zinc finger-like (FEZL) transcription factor, which was previously identified as a factor controlling mastitis resistance in the same half-sib families, bound the C stretch of IGF1R. The susceptible type of FEZL with a glycine stretch containing 13 glycines (13G) and the longer C stretch of IGF1R together enhanced expression of IGF1R. Enhancing IGF1R inhibited autophagy in response to Streptococcus agalactiae invasion of mammary epithelial cells, whereas treatment with rapamycin, a known inducer of autophagy, rescued it. Cows carrying the variant combination of 13GFEZL might be more susceptible to mastitis as the result of impaired autophagy. Our results suggest that IGF1R could control innate immunity in mammals and serve as a potential tool for preventing mastitis.

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

    PubMed

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

    2015-11-24

    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.

  1. 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

  2. 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

  3. The 3' Untranslated Region of the Cyclin B mRNA Is Not Sufficient to Enhance the Synthesis of Cyclin B during a Mitotic Block in Human Cells

    PubMed Central

    Schnerch, Dominik; Follo, Marie; Felthaus, Julia; Engelhardt, Monika; Wäsch, Ralph

    2013-01-01

    Antimitotic agents are frequently used to treat solid tumors and hematologic malignancies. However, one major limitation of antimitotic approaches is mitotic slippage, which is driven by slow degradation of cyclin B during a mitotic block. The extent to which cyclin B levels decline is proposed to be governed by an equilibrium between cyclin B synthesis and degradation. It was recently shown that the 3' untranslated region (UTR) of the murine cyclin B mRNA contributes to the synthesis of cyclin B during mitosis in murine cells. Using a novel live-cell imaging-based technique allowing us to study synthesis and degradation of cyclin B simultaneously at the single cell level, we tested here the role of the human cyclin B 3'UTR in regulating cyclin B synthesis during mitosis in human cells. We observed that the cyclin B 3'UTR was not sufficient to enhance cyclin B synthesis in human U2Os, HeLa or hTERT RPE-1 cells. A better understanding of how the equilibrium of cyclin B is regulated in mitosis may contribute to the development of improved therapeutic approaches to prevent mitotic slippage in cancer cells treated with antimitotic agents. PMID:24058555

  4. 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. PMID:26245723

  5. Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region.

    PubMed

    Cassetti, M C; Noah, D L; Montelione, G T; Krug, R M

    2001-10-25

    We test the hypothesis that the translation machinery in cells infected by influenza A virus efficiently translates only mRNAs that possess the influenza viral 5' untranslated region (5'-UTR) by introducing mRNAs directly into the cytoplasm of infected cells. This strategy avoids effects due to the inhibition of the nuclear export of cellular mRNAs mediated by the viral NS1 protein. In one approach, we transfect in vitro synthesized mRNAs into infected cells and demonstrate that these mRNAs are efficiently translated whether or not they possess the influenza viral 5'-UTR. In the second approach, an mRNA is synthesized endogenously in the cytoplasm of influenza A virus infected cells by a constitutively expressed T7 RNA polymerase. Although this mRNA is uncapped and lacks the influenza viral 5'-UTR sequence, it is efficiently translated in infected cells via an internal ribosome entry site. We conclude that the translation machinery in influenza A virus infected cells is capable of efficiently translating all mRNAs and that the switch from cellular to virus-specific protein synthesis that occurs during infection results from other processes.

  6. 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

  7. A highly conserved sequence in the 3'-untranslated region of the drosophila Adh gene plays a functional role in Adh expression.

    PubMed Central

    Parsch, J; Stephan, W; Tanda, S

    1999-01-01

    Phylogenetic analysis identified a highly conserved eight-base sequence (AAGGCTGA) within the 3'-untranslated region (UTR) of the Drosophila alcohol dehydrogenase gene, Adh. To examine the functional significance of this conserved motif, we performed in vitro deletion mutagenesis on the D. melanogaster Adh gene followed by P-element-mediated germline transformation. Deletion of all or part of the eight-base sequence leads to a twofold increase in in vivo ADH enzymatic activity. The increase in activity is temporally and spatially general and is the result of an underlying increase in Adh transcript. These results indicate that the conserved 3'-UTR motif plays a functional role in the negative regulation of Adh gene expression. The evolutionary significance of our results may be understood in the context of the amino acid change that produces the ADH-F allele and also leads to a twofold increase in ADH activity. While there is compelling evidence that the amino acid replacement has been a target of positive selection, the conservation of the 3'-UTR sequence suggests that it is under strong purifying selection. The selective difference between these two sequence changes, which have similar effects on ADH activity, may be explained by different metabolic costs associated with the increase in activity. PMID:9927459

  8. Succinate dehydrogenase b mRNA of Drosophila melanogaster has a functional iron-responsive element in its 5'-untranslated region.

    PubMed

    Kohler, S A; Henderson, B R; Kühn, L C

    1995-12-22

    Iron-responsive elements (IREs) are cis-acting mRNA stem-loop structures that specifically bind cytoplasmic iron regulatory proteins (IRPs). IRP-IRE interactions mediate the coordinate post-transcriptional regulation of key proteins in iron metabolism, such as ferritin, transferrin receptor, and erythroid 5-aminolevulinic acid synthase. Depending on whether the IRE is located in the 5'- or 3'-untranslated region (UTR), binding of IRP will inhibit mRNA translation or degradation, respectively. Here we describe a new IRE in the 5'-UTR of succinate dehydrogenase subunit b (SDHb) mRNA of Drosophila melanogaster. The SDHb IRE binds in vitro to vertebrate and insect IRPs with a high affinity equal to that of human ferritin H chain IRE. Under conditions of iron deprivation, SDHb mRNA of Drosophila SL-2 cells shifts to a non-polysome-bound pool. Moreover, translation of a human growth hormone mRNA with the SDHb IRE in its 5'-UTR is iron-dependent in stably transfected L cells. We conclude that the SDHb IRE mediates translational inhibition both in insect and vertebrate cells. This constitutes the first identification of a functional IRE in insects. Furthermore, Drosophila SDHb represents the second example, after porcine mitochondrial aconitase, of an enzyme of the citric acid cycle whose mRNA possesses all necessary features for translational regulation by cellular iron levels. PMID:8530520

  9. A variant within the AQP1 3'-untranslated region is associated with running performance, but not weight changes, during an Ironman Triathlon.

    PubMed

    Saunders, Colleen J; Posthumus, Michael; O'Connell, Kevin; September, Alison V; Collins, Malcolm

    2015-01-01

    The objective of this study was to test the association of the rs1049305 (G > C) variant within the 3'-untranslated region of the aquaporin 1 gene, AQP1, with changes in body weight, post-race serum sodium concentration and performance in Ironman triathletes. Five hundred and four male Ironman triathletes were genotyped for the rs1049305 variant within the AQP1 gene. Change in pre- and post-race body weight was calculated for 470 triathletes and used as a proxy for changes in body fluid during the race, as well as to divide triathletes into biologically relevant weight-loss groups (0-3%, 3-5% and >5%). There were no rs1049305 genotype effects on post-race serum sodium concentrations (P = 0.647), pre-race weight (P = 0.610) nor relative weight change during the Ironman Triathlons (P = 0.705). In addition, there were no significant differences in genotype (P = 0.640) nor allele (P = 0.643) distributions between the weight loss groups. However, triathletes who carry a C-allele were found to complete the 42.2-km run stage faster (mean 286, s = 49 min) than triathletes with a GG genotype (mean 296, s = 47 min; P = 0.032). The AQP1 rs1049305 variant is associated with running performance, but not relative body weight change, during the 2000, 2001 and 2006 South African Ironman Triathlons.

  10. Variant in the 5' untranslated region of insulin-like growth factor 1 receptor is associated with susceptibility to mastitis in cattle.

    PubMed

    Sugimoto, Mayumi; Sugimoto, Yoshikazu

    2012-09-01

    Mastitis is a common infectious disease of the mammary gland and generates large losses in the dairy industry. By means of positional cloning and functional analysis techniques, we here show that insulin-like growth factor 1 receptor (IGF1R) can possibly mediate susceptibility to mastitis through autophagy. Scanning the whole genome of cows (Bos taurus) that were susceptible or resistant to mastitis in the half-sib families revealed that susceptible cows had a relatively long stretch of cytosine residues (C stretch) in the 5' untranslated region of IGF1R. The forebrain embryonic zinc finger-like (FEZL) transcription factor, which was previously identified as a factor controlling mastitis resistance in the same half-sib families, bound the C stretch of IGF1R. The susceptible type of FEZL with a glycine stretch containing 13 glycines (13G) and the longer C stretch of IGF1R together enhanced expression of IGF1R. Enhancing IGF1R inhibited autophagy in response to Streptococcus agalactiae invasion of mammary epithelial cells, whereas treatment with rapamycin, a known inducer of autophagy, rescued it. Cows carrying the variant combination of 13GFEZL might be more susceptible to mastitis as the result of impaired autophagy. Our results suggest that IGF1R could control innate immunity in mammals and serve as a potential tool for preventing mastitis. PMID:22973545

  11. 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.

  12. 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.

  13. The effect of different 3' untranslated regions on the accumulation and stability of transcripts of a gfp transgene in chloroplasts of transplastomic tobacco.

    PubMed

    Tangphatsornruang, Sithichoke; Birch-Machin, Ian; Newell, Christine A; Gray, John C

    2011-07-01

    The 3' untranslated region (3' UTR) of transcripts is a major determinant of transcript stability in plastids and plays an important role in regulating gene expression. In order to compare the effect of different 3' UTRs on transgene expression in tobacco chloroplasts, the 3' UTRs from the tobacco chloroplast rbcL, psbA, petD and rpoA genes and the terminator region of the Escherichia coli rrnB operon were inserted downstream of the gfp reporter gene under the control of the psbA promoter, and the constructs were introduced into the plastid genome by particle bombardment. RNA-gel blot analysis of homoplasmic transplastomic plants identified gfp transcripts of ~1.0 and ~1.4 kb from all constructs and showed that plants expressing gfp with the rrnB terminator contained 4 times more gfp transcripts than plants expressing gfp with the rbcL and rpoA 3' UTRs. The amounts of transcripts accumulated roughly correlated with the half-life of the transcripts, determined by RNA-gel blot analysis of transcripts present in leaves treated with actinomycin D to prevent continued transcription of the chimeric gfp genes. Transcripts containing the 3' region of rrnB were most stable, with half-lives of ~43 h, considerably longer than the half-lives of the other ~1.0 kb gfp transcripts (13-26 h). Immunoblot analysis with antibodies to GFP indicated that all plants contained about the same amount of GFP (~0.2% total soluble protein), suggesting either that translation was limited by something other than the amount of transcript or that the 3' UTR was affecting translation.

  14. Identification of a novel mutation in the β-globin gene 3' untranslated region (HBB: c.*+118A > G) in Spain.

    PubMed

    Herrera, Maria Ascensión; De La Fuente-Gonzalo, Félix; González, Fernando Ataúlfo; Nieto, Jorge M; Dominguez, Alejandra Blum; Villegas, Ana; Ropero, Paloma

    2015-01-01

    The 3' untranslated region (3'UTR) region is well known to be associated with mRNA stability because of its associations with 3' end processing, polyadenylation and mRNA capping. Mutations located in this area cause a β-thalassemia (β-thal) phenotype compatible with β(+)-thal. Two brothers, 49- and 41-years-old, were diagnosed with β-thal intermedia (β-TI) at 2 years of age. The β-globin gene from the promoter region to the 3'UTR was sequenced and both brothers were diagnosed to be compound heterozygotes for the 3'UTR +1592 (A > G) (HBB: c.*+118A > G) and codon 39 (C > T) (HBB: c.118C > T) mutations. Their mother was a carrier of the nonsense codon 39 mutation and her hematological data suggested β-thal trait; their father was a carrier of the 3'UTR +1592 mutation, though he did not have hematological parameters associated with β-thal. The adenine at position +1592 or +118 bases downstream of the termination codon is highly conserved among primates and placental mammals, as it is located between the polyadenylation A signal (PAS) and the polyadenylation A cleavage (PAC) sites. Given its location, it is likely that this mutation would interfere with mRNA maturation; however, the clinical data of the heterozygous carriers show virtually no significant alterations. Therefore, we suggest that the impact on cleavage-stimulation factor (CstF) recognition of the mRNA sequence would be minimal and not significantly alter polyadenylation. Although the mechanism is not known, and because the carrier has no β-thal minor, the mRNA is stable enough that the synthesis of the β-globin chain is unaffected. PMID:25572186

  15. 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

  16. 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

  17. Identification of two proteins that bind to a pyrimidine-rich sequence in the 3'-untranslated region of GAP-43 mRNA.

    PubMed Central

    Irwin, N; Baekelandt, V; Goritchenko, L; Benowitz, L I

    1997-01-01

    GAP-43 is a membrane phosphoprotein that is important for the development and plasticity of neural connections. In undifferentiated PC12 pheochromocytoma cells, GAP-43 mRNA degrades rapidly ( t = 5 h), but becomes stable when cells are treated with nerve growth factor. To identify trans- acting factors that may influence mRNA stability, we combined column chromatography and gel mobility shift assays to isolate GAP-43 mRNA binding proteins from neonatal bovine brain tissue. This resulted in the isolation of two proteins that bind specifically and competitively to a pyrimidine-rich sequence in the 3'-untranslated region of GAP-43 mRNA. Partial amino acid sequencing revealed that one of the RNA binding proteins coincides with FBP (far upstream element binding protein), previously characterized as a protein that resembles hnRNP K and which binds to a single-stranded, pyrimidine-rich DNA sequence upstream of the c -myc gene to activate its expression. The other binding protein shares sequence homology with PTB, a polypyrimidine tract binding protein implicated in RNA splicing and regulation of translation initiation. The two proteins bind to a 26 nt pyrimidine-rich sequence lying 300 nt downstream of the end of the coding region, in an area shown by others to confer instability on a reporter mRNA in transient transfection assays. We therefore propose that FBP and the PTB-like protein may compete for binding at the same site to influence the stability of GAP-43 mRNA. PMID:9092640

  18. 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.

  19. The 3' untranslated region of the hsp 70 genes maintains the level of steady state mRNA in Trypanosoma brucei upon heat shock.

    PubMed Central

    Lee, M G

    1998-01-01

    An increase in the transcriptional efficiency at elevated temperatures is a characteristic of transcription of heat shock protein (hsp) coding genes in most eukaryotes analyzed to date. The regulatory mechanism for hsp 70 genes expression in Trypanosoma brucei does not follow the conventional transcriptional induction mechanism. The hsp 70 locus of T.brucei appears in a permanently activated state, and transcriptional induction of hsp 70 genes by heat shock does not occur in this organism. Therefore, the differential expression of the hsp 70 genes in trypanosomes is, to a large extent, post-transcriptionally controlled. Mechanisms of post-transcriptional control of the hsp 70 gene expression were investigated. Procyclic trypanosomes were normally maintained at approximately 25 degreesC. Incubation of procyclic trypanosomes at 41 degreesC drastically reduced the steady state mRNA levels of many protein coding genes. In contrast, the expression of the hsp 70 genes is either maintained at a high level or is up-regulated. The hsp 70 intergenic region promoter together with its 3' splice acceptor sites and the 5' untranslated region (UTR) are not sufficient to maintain or up-regulate the mRNA level of a reporter gene upon heat shock. However, addition of the 3' UTR of hsp 70 genes to a reporter gene, driven by different promoters, maintained a high level expression of the mRNA during heat shock. These results suggested that the 3' UTR of the hsp 70 genes is primarily responsible for the maintenance of mRNA level during heat shock, while mRNA containing the 3' UTR from many other genes may be rapidly degraded by heat shock induced processes. PMID:9705515

  20. 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.

  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. Structure of the mouse leukaemia inhibitory factor receptor gene: regulated expression of mRNA encoding a soluble receptor isoform from an alternative 5' untranslated region.

    PubMed Central

    Chambers, I; Cozens, A; Broadbent, J; Robertson, M; Lee, M; Li, M; Smith, A

    1997-01-01

    The low-affinity leukaemia inhibitory factor receptor (LIF-R) is a component of cell-surface receptor complexes for the multifunctional cytokines leukaemia inhibitory factor, ciliary neurotrophic factor, oncostatin M and cardiotrophin-1. Both soluble and transmembrane forms of the protein have been described and several LIF-R mRNAs have been reported previously. In order to determine the coding potential of LIF-R mRNAs we have isolated and characterized the mouse LIF-R gene. mRNA encoding soluble LIF-R (sLIF-R) is formed by inclusion of an exon in which polyadenylation signals are provided by a B2 repeat. This exon is located centrally within the LIF-R gene but is excluded from the transmembrane LIF-R mRNA by alternative splicing. The transmembrane receptor is encoded by 19 exons distributed over 38 kb. Two distinct 5' non-coding exons have been identified, indicating the existence of alternative promoters. One of these is G/C rich and possesses a consensus initiator sequence as well as potential Sp1 binding sites. Expression of exon 1 from this promoter occurs in a wide variety of tissues, whereas expression of the alternative 5' untranslated region (exon 1a) is normally restricted to liver, the principal source of sLIF-R. During pregnancy expression of exon 1a becomes detectable also in the uterus. Expression of exon 1a increases dramatically during gestation and is accompanied by a similar quantitative rise in expression of sLIF-R mRNA. These findings establish that expression of LIF-R is under complex transcriptional control and indicate that regulated expression of the soluble cytokine receptor isoform may be due principally to an increase in the activity of a dedicated promoter. PMID:9396734

  3. Poly(A)-Binding Protein Facilitates Translation of an Uncapped/Nonpolyadenylated Viral RNA by Binding to the 3′ Untranslated Region

    PubMed Central

    Iwakawa, Hiro-oki; Tajima, Yuri; Taniguchi, Takako; Kaido, Masanori; Mise, Kazuyuki; Tomari, Yukihide; Taniguchi, Hisaaki

    2012-01-01

    Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5′ cap and a 3′ poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3′ untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3′CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3′CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3′CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3′ UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3′CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3′CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3′ UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3′CITE as substitutes for the 3′ poly(A) tail and the 5′ cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA. PMID:22593149

  4. 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. PMID:25997640

  5. A SNP in the 3'-untranslated region of AMPKγ1 may associate with serum ketone body and milk production of Holstein dairy cows.

    PubMed

    Mahmoudi, Ahmad; Zargaran, Amir; Amini, Hamid-Reza; Assadi, Assad; Vajdi Hokmabad, Reza; Eghbalsaied, Shahin

    2015-12-10

    AMPK is the key switch for providing the energy balance between cellular anabolic and catabolic processes. In this study, we aimed to screen the PRKAG1 (AMPKγ1) gene in high, moderate, and low producing Holstein dairy cows. A sample of 100 pregnant dairy cows, comprising 41 high, 33 moderate, and 26 low milk yields were selected from three large dairy herds in Isfahan province of Iran. Body condition score (BCS) was estimated before parturition while beta hydroxyl butyric acid (BHBA) as a measure of ketone bodies was measured at the fifth day postpartum. In addition, using three primer pairs covering exons 2-11 and 3'-UTR of the PRKAG1 gene, a random sample of 10 high milk yield dairy cows were amplified and sequenced. The sequencing results showed the presence of a T12571C mutation in intron 6 and a T14280C mutation in the 3'-untranslated region (UTR) of the PRKAG1 gene. Following a PCR reaction for amplification of the 3'-UTR amplicons, single strand conformation polymorphism (SSCP) assay was implemented for discrimination of the mutation in the studied population. Then, we evaluated if the mutation associates with the BCS, serum BHBA level, and production traits. The experimental analysis showed that the mutated allele significantly increased the BHBA level, BCS, as well as milk and protein yield. Bioinformatic study revealed that this 3'-UTR mutation distorts the target site of mir-423-5p microRNA which is one of the most highly expressed microRNAs in the bovine mammary gland, liver, and kidney. Given the role of AMPK in energy metabolism, the newly identified 3'-UTR mutation highlights the importance of AMPK and suggests a role of miRNAs for regulation of cellular metabolism, metabolism disorders, and production traits in Holstein dairy cows. PMID:26226224

  6. 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

  7. A SNP in the 3'-untranslated region of AMPKγ1 may associate with serum ketone body and milk production of Holstein dairy cows.

    PubMed

    Mahmoudi, Ahmad; Zargaran, Amir; Amini, Hamid-Reza; Assadi, Assad; Vajdi Hokmabad, Reza; Eghbalsaied, Shahin

    2015-12-10

    AMPK is the key switch for providing the energy balance between cellular anabolic and catabolic processes. In this study, we aimed to screen the PRKAG1 (AMPKγ1) gene in high, moderate, and low producing Holstein dairy cows. A sample of 100 pregnant dairy cows, comprising 41 high, 33 moderate, and 26 low milk yields were selected from three large dairy herds in Isfahan province of Iran. Body condition score (BCS) was estimated before parturition while beta hydroxyl butyric acid (BHBA) as a measure of ketone bodies was measured at the fifth day postpartum. In addition, using three primer pairs covering exons 2-11 and 3'-UTR of the PRKAG1 gene, a random sample of 10 high milk yield dairy cows were amplified and sequenced. The sequencing results showed the presence of a T12571C mutation in intron 6 and a T14280C mutation in the 3'-untranslated region (UTR) of the PRKAG1 gene. Following a PCR reaction for amplification of the 3'-UTR amplicons, single strand conformation polymorphism (SSCP) assay was implemented for discrimination of the mutation in the studied population. Then, we evaluated if the mutation associates with the BCS, serum BHBA level, and production traits. The experimental analysis showed that the mutated allele significantly increased the BHBA level, BCS, as well as milk and protein yield. Bioinformatic study revealed that this 3'-UTR mutation distorts the target site of mir-423-5p microRNA which is one of the most highly expressed microRNAs in the bovine mammary gland, liver, and kidney. Given the role of AMPK in energy metabolism, the newly identified 3'-UTR mutation highlights the importance of AMPK and suggests a role of miRNAs for regulation of cellular metabolism, metabolism disorders, and production traits in Holstein dairy cows.

  8. A single nucleotide polymorphism in the 3'-untranslated region of the KRAS gene disrupts the interaction with let-7a and enhances the metastatic potential of osteosarcoma cells.

    PubMed

    Zhang, Shiqian; Hou, Chunying; Li, Guojun; Zhong, Yang; Zhang, Jie; Guo, Xinzhen; Li, Baoxin; Bi, Zhenggang; Shao, Ming

    2016-09-01

    The objective of the present study was to explore the molecular mechanism with which a single nucleotide polymorphism (rs61764370) interferes with the interaction between the 3'-untranslated region (3'-UTR) of Kirsten rat sarcoma viral oncogene homolog (KRAS) and let-7a, and its association with the metastasis of osteosarcoma (OS). In this study, we confirmed that KRAS is a target of let-7a in OS cells, and the introduction of rs61764370 minor allele into KRAS 3'-UTR significantly compromised the microRNA (miRNA)/mRNA interaction using a luciferase reporter system. Additionally, a total of 36 OS tissue samples of three different genotypes (TT,22; TG,10; GG,4) were obtained, and the expression of let-7a and KRAS was determined. We showed that let-7a mRNA expression was similar between each group whereas the mRNA and protein expression of KRAS in the TT genotype group was significantly lower than that in the GT or GG genotype groups. Moreover, we identified a negative regulatory relationship between let-7a and KRAS. Furthermore, we demonstrated that let-7a and KRAS interfered with the viability, invasiveness and migration of OS cells genotyped as TT. In the OS cells genotyped as TG, let-7a exerted minimal effects, and the effect of KRAS siRNA remained. Taken together, the findings of the present study demonstrated that the KRAS 3'-UTR rs61764370 polymorphism interfered with miRNA/mRNA interaction, and showed that the minor allele was associated with an elevated risk of developing metastatic disease in OS. PMID:27430246

  9. Multiple, conserved iron-responsive elements in the 3'-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2.

    PubMed

    Erlitzki, Ronit; Long, Joanne C; Theil, Elizabeth C

    2002-11-01

    Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.

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

    PubMed

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

    2006-03-01

    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.

  11. 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

  12. 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

  13. 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

  14. Association of HLA-G 3’ Untranslated Region Polymorphisms with Systemic Lupus Erythematosus in a Japanese Population: A Case-Control Association Study

    PubMed Central

    Hachiya, Yuki; Kawasaki, Aya; Oka, Shomi; Kondo, Yuya; Ito, Satoshi; Matsumoto, Isao; Kusaoi, Makio; Amano, Hirofumi; Suda, Akiko; Setoguchi, Keigo; Nagai, Tatsuo; Shimada, Kota; Sugii, Shoji; Okamoto, Akira; Chiba, Noriyuki; Suematsu, Eiichi; Ohno, Shigeru; Katayama, Masao; Kono, Hajime; Hirohata, Shunsei; Takasaki, Yoshinari; Hashimoto, Hiroshi; Sumida, Takayuki; Nagaoka, Shouhei; Tohma, Shigeto; Furukawa, Hiroshi

    2016-01-01

    HLA-G plays a role in fetal-maternal tolerance as well as immunoregulation, and has been suggested to be involved in autoimmune diseases and cancers. HLA-G encodes two potentially functional polymorphisms in the 3’ untranslated region, 14bp insertion/deletion (14bp indel, rs371194629) and a single nucleotide polymorphism rs1063320, previously reported to affect HLA-G expression level or splicing isoform and to be associated with susceptibility to systemic lupus erythematosus (SLE). However, the results of SLE association studies are inconsistent, probably due to the small sample size of each study and lack of consideration of linkage disequilibrium (LD) with HLA-class II haplotypes in each population. In this study, we performed association studies of these polymorphisms on 843 patients with SLE and 778 healthy controls in a Japanese population, in many of whom HLA-DRB1 alleles have been genotyped at the four-digit level. LD was detected between DRB1*13:02, protective against multiple autoimmune diseases in the Japanese, and the rs1063320 G (D’ = 0.86, r2 = 0.02) and with 14bp del (D’ = 0.62, r2 = 0.01), but not between SLE-susceptible DRB1*15:01 and HLA-G. Although significant association with overall SLE was not detected, 14bp ins allele was significantly associated with SLE with the age of onset <20 years, when compared with healthy controls (P = 0.0067, PFDR = 0.039, OR 1.44, additive model) or with SLE patients with the age of onset ≥20 (P = 0.033, PFDR = 0.0495, OR 2.09, additive model). This association remained significant after conditioning on DRB1*13:02 or DRB1*15:01. On the other hand, significant association was detected between rs1063320 C and anti-RNP antibody and anti-Sm antibody positive SLE, which was dependent on negative LD with DRB1*13:02. eQTL analysis showed reduced HLA-G mRNA level in 14bp ins/ins individuals. In conclusion, our observations showed that HLA-G 14bp ins allele represents a genetic contribution on early-onset SLE

  15. Association of HLA-G 3' Untranslated Region Polymorphisms with Systemic Lupus Erythematosus in a Japanese Population: A Case-Control Association Study.

    PubMed

    Hachiya, Yuki; Kawasaki, Aya; Oka, Shomi; Kondo, Yuya; Ito, Satoshi; Matsumoto, Isao; Kusaoi, Makio; Amano, Hirofumi; Suda, Akiko; Setoguchi, Keigo; Nagai, Tatsuo; Shimada, Kota; Sugii, Shoji; Okamoto, Akira; Chiba, Noriyuki; Suematsu, Eiichi; Ohno, Shigeru; Katayama, Masao; Kono, Hajime; Hirohata, Shunsei; Takasaki, Yoshinari; Hashimoto, Hiroshi; Sumida, Takayuki; Nagaoka, Shouhei; Tohma, Shigeto; Furukawa, Hiroshi; Tsuchiya, Naoyuki

    2016-01-01

    HLA-G plays a role in fetal-maternal tolerance as well as immunoregulation, and has been suggested to be involved in autoimmune diseases and cancers. HLA-G encodes two potentially functional polymorphisms in the 3' untranslated region, 14bp insertion/deletion (14bp indel, rs371194629) and a single nucleotide polymorphism rs1063320, previously reported to affect HLA-G expression level or splicing isoform and to be associated with susceptibility to systemic lupus erythematosus (SLE). However, the results of SLE association studies are inconsistent, probably due to the small sample size of each study and lack of consideration of linkage disequilibrium (LD) with HLA-class II haplotypes in each population. In this study, we performed association studies of these polymorphisms on 843 patients with SLE and 778 healthy controls in a Japanese population, in many of whom HLA-DRB1 alleles have been genotyped at the four-digit level. LD was detected between DRB1*13:02, protective against multiple autoimmune diseases in the Japanese, and the rs1063320 G (D' = 0.86, r2 = 0.02) and with 14bp del (D' = 0.62, r2 = 0.01), but not between SLE-susceptible DRB1*15:01 and HLA-G. Although significant association with overall SLE was not detected, 14bp ins allele was significantly associated with SLE with the age of onset <20 years, when compared with healthy controls (P = 0.0067, PFDR = 0.039, OR 1.44, additive model) or with SLE patients with the age of onset ≥20 (P = 0.033, PFDR = 0.0495, OR 2.09, additive model). This association remained significant after conditioning on DRB1*13:02 or DRB1*15:01. On the other hand, significant association was detected between rs1063320 C and anti-RNP antibody and anti-Sm antibody positive SLE, which was dependent on negative LD with DRB1*13:02. eQTL analysis showed reduced HLA-G mRNA level in 14bp ins/ins individuals. In conclusion, our observations showed that HLA-G 14bp ins allele represents a genetic contribution on early-onset SLE independent

  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. Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation.

    PubMed

    Zhu, Hui; Duan, Cheng-Guo; Hou, Wei-Na; Du, Quan-Sheng; Lv, Dian-Qiu; Fang, Rong-Xiang; Guo, Hui-Shan

    2011-12-01

    RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.

  18. 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 Central

    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

  19. microRNA-18b modulates insulin-like growth factor-1 expression in deer antler cell proliferation by directly targeting its 3' untranslated region.

    PubMed

    Hu, Wei; Li, Mu; Hu, Rui; Li, Ting; Meng, Xingyu

    2015-04-01

    Insulin-like growth factor-1 (IGF-1) is a multipromoter gene that has complex biological functions and plays an important role in Chinese sika deer antler cell differentiation and proliferation. microRNAs and their roles in deer antler growth have attracted much attention. In the present study, to investigate the effect of microRNAs on the regulation of IGF-1 during the rapid growth of antlers, miRNA GeneChip analysis and TargetScan Human software were used to screen microRNAs that bind to the 3' untranslated region (3'UTR) of IGF-1. The results indicated that a significantly differential expression of miR-18b was observed in cartilage and mesenchymal of antler tip tissue and the presence of miR-18b-binding sites within the IGF-1 3'UTR. A miR-18b mimic was then transfected into antler cartilage cells to overexpress miR-18b and the expression levels were quantified by real-time PCR. Real-time PCR showed that the expression level of miR-18b in transfected cells was significantly increased compared with the control group (p<0.01). Dual luciferase assays revealed that miR-18b decreased the fluorescence value of the luciferase reporter gene in the group transfected with the wild-type vector of IGF-1 3'UTR. In contrast, the relative luciferase activity in the group transfected with the mutant vector of IGF-1 3'UTR did not change obviously. MTT assays and cell cycle analyses confirmed that overexpression of the miR-18b mimic inhibited the proliferation of cartilage cells. In contrast, transfection of a miR-18b inhibitor increased the cell proliferation rate. Furthermore, Western blot analyses revealed that overexpression of miR-18b mimics downregulated the protein levels of IGF-1, while IGF-1 expression increased after transfection of miR-18b inhibitors. Taken together, our findings show that miR-18b is a potentially novel target in deer antler cell proliferation. miR-18b may modulate IGF-1 expression of sika deer antler. PMID:25756952

  20. Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region

    PubMed Central

    Chattopadhyay, Maitreyi; Stupina, Vera A.; Gao, Feng; Szarko, Christine R.; Kuhlmann, Micki M.; Yuan, Xuefeng; Shi, Kerong

    2015-01-01

    ABSTRACT Turnip crinkle virus (TCV) contains a structured 3′ region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3′ untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3′UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. IMPORTANCE Genomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions

  1. 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

  2. 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

  3. Protein kinase A stimulates binding of multiple proteins to a U-rich domain in the 3'-untranslated region of lactate dehydrogenase A mRNA that is required for the regulation of mRNA stability.

    PubMed

    Tian, D; Huang, D; Brown, R C; Jungmann, R A

    1998-10-23

    We have explored the molecular basis of the cAMP-induced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasmic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-nucleotide uridine-rich sequence in the LDH 3'-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that specific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stranded stem structure, thus indicating a high degree of primary and secondary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and contains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. The 3'-untranslated region binding activity of the proteins is up-regulated after protein kinase A activation, whereas protein dephosphorylation is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and CSR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processing pathway that specifically stabilizes LDH mRNA in response to activation of the protein kinase A signal transduction pathway.

  4. The length of the combined 3' untranslated region and poly(A) tail does not control rates of glyceraldehyde-3-phosphate dehydrogenase mRNA translation in three species of parasitic protists.

    PubMed

    ter Kuile, B H; Sallés, F J

    2000-06-01

    Experimental observations suggested that the length of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA 3' end has a role in regulating rates of translation in the parasitic protists Trypanosoma brucei, Leishmania donovani, and Trichomonas vaginalis. Using a PCR assay for poly(A) tail length, we measured the size of the RNA 3' end under different growth conditions in all three species. Our results showed that the combined 3' untranslated region and poly(A) tail of GAPDH mRNA do not vary with different rates of translation.

  5. Hepatitis C Genotype Prevalence in Monastir Region, Tunisia: Correlation between 5' Untranslated Region (5'UTR), Non-structural 5B (NS5B), and Core Sequences in HCV Subtyping.

    PubMed

    Souii, Amira; Elargoubi, Aida; Fallecker, Catherine; Mastouri, Maha; Drouet, Emmanuel

    2016-09-01

    Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. It constitutes a major public health around the world. There is no vaccine available against HCV, and current therapies are effective in only small percentage of patients. HCV has wide population-specific genotype variability. Genotype knowledge and viral load assessment are equally important for designing therapeutic strategies. Taking into account that the molecular epidemiology of HCV variants circulating in Tunisia is not yet well elucidated, and that, at present, little is known about the distribution pattern of HCV in Monastir region (Tunisia), we aimed, herein, to evaluate the prevalence of HCV genotypes in Monastir and to identify risk-related factors. For this purpose, 50 anti-HCV antibody-positive cases were diagnosed and subjected to viral RNA extraction, amplification, genotyping, and viral load quantification. Molecular epidemiology was studied by 5' untranslated region (5' UTR) sequencing as compared with the non-structural 5B (NS5B) and core region sequences. Overall concordance between 5' UTR, core, and NS5B sequencing was 100 %. The highest prevalent genotype was 1b (50 %) followed by genotypes 1a (16 %), 4a (12 %), 2a (10 %), 2c (8 %), and 3a (4 %). Interestingly, the subtype 1b had a statistically significant higher viral load than the other genotypes followed by subtype 1a. Based on these data, this study revealed a high prevalence of HCV genotype 1 (subtypes 1b and 1a) compared to other genotypes. A continued monitoring of HCV and knowledge of circulating genotypes could impact on future vaccine formulations. PMID:27189386

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. A polymorphism at the microRNA binding site in the 3′-untranslated region of C14orf101 is associated with the risk of gastric cancer development

    PubMed Central

    Wang, Cuiju; Zhao, Yufei; Ming, Yanming; Zhao, Shengnan; Guo, Zhanjun

    2016-01-01

    MicroRNAs (miRNAs) bind to the 3′-untranslated regions (3′-UTRs) of mRNAs, affecting translation and regulating cell differentiation, tumorigenesis and apoptosis. Genetic polymorphisms in these regions in target genes are able to affect the binding affinity between miRNA and target genes, ultimately affecting the expression of individual miRNAs. In the present case-control study, genotyping of 5 microRNA single nucleotide polymorphisms (SNPs) located at the binding site of the 3′-UTR of RYR3 (rs1044129), C14orf101 (rs4901706), KIAA0423 (rs1053667), GOLGA7 (rs11337) and KRT81 (rs3660) genes was assessed in order to investigate its role in gastric cancer (GC). The results indicated that the rs4901706 SNP, which is located in the 3′-UTR of C14orf101, was associated with GC development risk, as determined by χ2 analysis (relative risk, 1.630; 95% confidence interval, 1.070–2.483; P=0.022). A Renilla/luciferase reporter assay also indicated the different binding affinity between the SNP of rs4901706 and microRNA. In conclusion, rs4901706 SNP of C14orf101 gene in the microRNA binding site may be used as a valuable biomarker when predicting GC risk. PMID:27602096

  14. A polymorphism at the microRNA binding site in the 3′-untranslated region of C14orf101 is associated with the risk of gastric cancer development

    PubMed Central

    Wang, Cuiju; Zhao, Yufei; Ming, Yanming; Zhao, Shengnan; Guo, Zhanjun

    2016-01-01

    MicroRNAs (miRNAs) bind to the 3′-untranslated regions (3′-UTRs) of mRNAs, affecting translation and regulating cell differentiation, tumorigenesis and apoptosis. Genetic polymorphisms in these regions in target genes are able to affect the binding affinity between miRNA and target genes, ultimately affecting the expression of individual miRNAs. In the present case-control study, genotyping of 5 microRNA single nucleotide polymorphisms (SNPs) located at the binding site of the 3′-UTR of RYR3 (rs1044129), C14orf101 (rs4901706), KIAA0423 (rs1053667), GOLGA7 (rs11337) and KRT81 (rs3660) genes was assessed in order to investigate its role in gastric cancer (GC). The results indicated that the rs4901706 SNP, which is located in the 3′-UTR of C14orf101, was associated with GC development risk, as determined by χ2 analysis (relative risk, 1.630; 95% confidence interval, 1.070–2.483; P=0.022). A Renilla/luciferase reporter assay also indicated the different binding affinity between the SNP of rs4901706 and microRNA. In conclusion, rs4901706 SNP of C14orf101 gene in the microRNA binding site may be used as a valuable biomarker when predicting GC risk.

  15. 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.

  16. 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

  17. 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 Central

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

    2011-01-01

    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. PMID:21389125

  18. A polymorphism in the 3′-untranslated region of the NPM1 gene causes illegitimate regulation by microRNA-337-5p and correlates with adverse outcome in acute myeloid leukemia

    PubMed Central

    Cheng, Chi Keung; Kwan, Tsz Ki; Cheung, Chi Ying; Ng, Kitty; Liang, Pei; Cheng, Suk Hang; Chan, Natalie P. H.; Ip, Rosalina K. L.; Wong, Raymond S. M.; Lee, Vincent; Li, Chi Kong; Yip, Sze Fai; Ng, Margaret H. L.

    2013-01-01

    Nucleophosmin, encoded by NPM1, is a haploinsufficient suppressor in hematologic malignancies. NPM1 mutations are mostly found in acute myeloid leukemia patients with normal karyotype and associated with favorable prognosis. A polymorphic nucleotide T deletion with unknown significance is present in the NPM1 3′-untranslated region. Here, we showed that the homozygous nucleotide T deletion was associated with adverse outcomes and could independently predict shortened survival in patients with de novo acute myeloid leukemia. Mechanistically, we demonstrated that the nucleotide T deletion created an illegitimate binding NPM1 for miR-337-5p, which was widely expressed in different acute myeloid leukemia subtypes and inhibited NPM1 expression. Accordingly, NPM1 levels were found to be significantly reduced and correlated with miR-337-5p levels in patients carrying a homozygous nucleotide T-deletion genotype. Together, our findings uncover a microRNA-mediated control of NPM1 expression that contributes to disease heterogeneity and suggest additional prognostic values of NPM1 in acute myeloid leukemia. PMID:23065518

  19. 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

  20. 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

  1. 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

  2. The Rev protein of human immunodeficiency virus type 1 counteracts the effect of an AU-rich negative element in the human papillomavirus type 1 late 3' untranslated region.

    PubMed Central

    Tan, W; Schwartz, S

    1995-01-01

    We have identified a sequence in the late 3' untranslated region of human papillomavirus type 1 mRNAs that acts posttranscriptionally to repress gene expression. Deletion analysis localized the inhibitory element to an AU-rich sequence between nucleotides 6958 and 6984 on the human papillomavirus type 1 genome. This sequence inhibits gene expression in an orientation-dependent manner. Upon transfection of eucaryotic cells with plasmids containing this sequence, approximately 4-fold-lower cytoplasmic mRNA levels and 64- to 128-fold-lower protein levels were produced compared with those produced by plasmids lacking the inhibitory sequence. Interestingly, providing the constitutive transport element of simian retrovirus type 1 in sense orientation counteracted inhibition exerted by the human papillomavirus type 1 sequence. Inhibition could also be overcome by the presence of human immunodeficiency virus type 1 Rev protein in trans and its target sequence, the Rev-responsive element, in cis. Rev is a nuclear protein and acts by promoting nuclear export of human immunodeficiency virus type 1 mRNAs encoding structural proteins. Our results are consistent with a model for human papillomavirus type 1 late-gene expression in which mRNAs containing human papillomavirus type 1 inhibitory sequences enter a nonproductive route in the nucleus, resulting in inefficient mRNA utilization. Rev directs mRNA containing inhibitory sequences to a productive route by interacting with the Rev-responsive element. PMID:7707519

  3. 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

  4. 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

  5. Ribosomal Protein S1 Specifically Binds to the 5′ Untranslated Region of the Pseudomonas aeruginosa Stationary-Phase Sigma Factor rpoS mRNA in the Logarithmic Phase of Growth

    PubMed Central

    Ševo, Milica; Buratti, Emanuele; Venturi, Vittorio

    2004-01-01

    The rpoS gene encodes the stationary-phase sigma factor (RpoS or σs), which was identified in several gram-negative bacteria as a central regulator controlling the expression of genes involved in cell survival in response to cessation of growth (stationary phase) and providing cross-protection against various stresses. In Pseudomonas aeruginosa, the levels of σs increase dramatically at the onset of the stationary phase and are regulated at the transcriptional and posttranscriptional levels. The P. aeruginosa rpoS gene is transcribed as a monocistronic rpoS mRNA transcript comprised of an unusually long 373-bp 5′ untranslated region (5′ UTR). In this study, the 5′ UTR and total protein extracts from P. aeruginosa logarithmic and stationary phases of growth were used in order to investigate the protein-RNA interactions that may modulate the translational process. It was observed that a 69-kDa protein, which corresponded to ribosomal protein S1, preferentially binds the 5′ UTR of the rpoS mRNA in the logarithmic phase and not in the stationary phase. This is the first report of a protein-rpoS mRNA 5′ UTR interaction in P. aeruginosa, and the possible involvement of protein S1 in translation regulation of rpoS is discussed. PMID:15262927

  6. 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

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

    2016-04-01

    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

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

    SciTech Connect

    Ally, A.; Nisson, P.E.

    1994-09-01

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

  8. The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations.

    PubMed

    Diederichs, Sven; Bartsch, Lorenz; Berkmann, Julia C; Fröse, Karin; Heitmann, Jana; Hoppe, Caroline; Iggena, Deetje; Jazmati, Danny; Karschnia, Philipp; Linsenmeier, Miriam; Maulhardt, Thomas; Möhrmann, Lino; Morstein, Johannes; Paffenholz, Stella V; Röpenack, Paula; Rückert, Timo; Sandig, Ludger; Schell, Maximilian; Steinmann, Anna; Voss, Gjendine; Wasmuth, Jacqueline; Weinberger, Maria E; Wullenkord, Ramona

    2016-01-01

    Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor-specific mutations not only in protein-coding sequences but also in non-coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this "dark matter" of the genome. Malignancy-driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5'-UTR and 3'-UTR Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non-coding RNA, such as microRNA, lncRNA, and lincRNA A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR-21 as well as tumor suppressor genes such as TP53/p53, APC, BRCA1, or RB1 can be affected by these alterations. In summary, coding-independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non-coding or allegedly silent mutations in tumorigenesis.

  9. Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions.

    PubMed Central

    Damgaard, C K; Dyhr-Mikkelsen, H; Kjems, J

    1998-01-01

    Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context. PMID:9685481

  10. The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations.

    PubMed

    Diederichs, Sven; Bartsch, Lorenz; Berkmann, Julia C; Fröse, Karin; Heitmann, Jana; Hoppe, Caroline; Iggena, Deetje; Jazmati, Danny; Karschnia, Philipp; Linsenmeier, Miriam; Maulhardt, Thomas; Möhrmann, Lino; Morstein, Johannes; Paffenholz, Stella V; Röpenack, Paula; Rückert, Timo; Sandig, Ludger; Schell, Maximilian; Steinmann, Anna; Voss, Gjendine; Wasmuth, Jacqueline; Weinberger, Maria E; Wullenkord, Ramona

    2016-01-01

    Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor-specific mutations not only in protein-coding sequences but also in non-coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this "dark matter" of the genome. Malignancy-driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5'-UTR and 3'-UTR Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non-coding RNA, such as microRNA, lncRNA, and lincRNA A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR-21 as well as tumor suppressor genes such as TP53/p53, APC, BRCA1, or RB1 can be affected by these alterations. In summary, coding-independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non-coding or allegedly silent mutations in tumorigenesis. PMID:26992833

  11. 5′-Untranslated Region of the Tryptophan Hydroxylase-2 Gene Harbors an Asymmetric Bidirectional Promoter but not Internal Ribosome Entry Site in vitro

    PubMed Central

    Chen, Guo-Lin; Miller, Gregory M.

    2009-01-01

    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. PMID:19344641

  12. The 3′-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA

    PubMed Central

    GOLDEN, DANIEL E.; HAJDUK, STEPHEN L.

    2005-01-01

    RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridines are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is specifically cleaved, uridines are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, noncoding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of the their cognate mRNA and anneal to form a binary RNA complex . An exception for this process may be cytochrome oxidase subunit II (COII) mRNA since a gene encoding a trans acting gRNA has not been identified. Using an in vitro editing assay we find that the 3′ UTR of COII, indeed, functions as a guide for both the site and number of uridines added to the coding region of the COII mRNA. We further show that the guiding sequence within the COII 3′ UTR can only function in COII editing when contiguous with the editing substrate, indicating that the 3′ UTR of COII lacks sequence or structure information necessary to function as a trans-acting gRNA. While other RNAs have been shown to “guide” RNA processing reactions, our discovery that the COII 3′ UTR directs editing of its cognate mRNA in cis, is a unique function for a 3′ UTR. The findings described here have led us to propose a new model for the evolution of gRNAs in kinetoplastids. PMID:15574518

  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. Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm ▿ †

    PubMed Central

    Lale, Rahmi; Berg, Laila; Stüttgen, Friederike; Netzer, Roman; Stafsnes, Marit; Brautaset, Trygve; Vee Aune, Trond Erik; Valla, Svein

    2011-01-01

    The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5′ untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (Pantitet), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C50 carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions. PMID:21335387

  15. Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells.

    PubMed

    Iwai, Y; Bickel, M; Pluznik, D H; Cohen, R B

    1991-09-25

    Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region. PMID:1917935

  16. Variants in the 3′ untranslated region of the KCNQ1-encoded Kv7.1 potassium channel modify disease severity in patients with type 1 long QT syndrome in an allele-specific manner

    PubMed Central

    Amin, Ahmad S.; Giudicessi, John R.; Tijsen, Anke J.; Spanjaart, Anne M.; Reckman, Yolan J.; Klemens, Christine A.; Tanck, Michael W.; Kapplinger, Jamie D.; Hofman, Nynke; Sinner, Moritz F.; Müller, Martina; Wijnen, Wino J.; Tan, Hanno L.; Bezzina, Connie R.; Creemers, Esther E.; Wilde, Arthur A. M.; Ackerman, Michael J.; Pinto, Yigal M.

    2012-01-01

    Aims Heterozygous mutations in KCNQ1 cause type 1 long QT syndrome (LQT1), a disease characterized by prolonged heart rate-corrected QT interval (QTc) and life-threatening arrhythmias. It is unknown why disease penetrance and expressivity is so variable between individuals hosting identical mutations. We aimed to study whether this can be explained by single nucleotide polymorphisms (SNPs) in KCNQ1's 3′ untranslated region (3′UTR). Methods and results This study was performed in 84 LQT1 patients from the Academic Medical Center in Amsterdam and validated in 84 LQT1 patients from the Mayo Clinic in Rochester. All patients were genotyped for SNPs in KCNQ1's 3′UTR, and six SNPs were found. Single nucleotide polymorphisms rs2519184, rs8234, and rs10798 were associated in an allele-specific manner with QTc and symptom occurrence. Patients with the derived SNP variants on their mutated KCNQ1 allele had shorter QTc and fewer symptoms, while the opposite was also true: patients with the derived SNP variants on their normal KCNQ1 allele had significantly longer QTc and more symptoms. Luciferase reporter assays showed that the expression of KCNQ1's 3′UTR with the derived SNP variants was lower than the expression of the 3′UTR with the ancestral SNP variants. Conclusion Our data indicate that 3′UTR SNPs potently modify disease severity in LQT1. The allele-specific effects of the SNPs on disease severity and gene expression strongly suggest that they are functional variants that directly alter the expression of the allele on which they reside, and thereby influence the balance between proteins stemming from either the normal or the mutant KCNQ1 allele. PMID:22199116

  17. Translation of a testis-specific Cu/Zn superoxide dismutase (SOD-1) mRNA is regulated by a 65-kilodalton protein which binds to its 5' untranslated region.

    PubMed Central

    Gu, W; Hecht, N R

    1996-01-01

    Mouse testes contain two distinct superoxide dismutase (SOD-1) transcripts which differ by 114 nucleotides in their 5' untranslated regions (UTRs) (W. Gu, C. Morales, and N. B. Hecht, J. Biol. Chem. 270:236-243, 1995). The shorter SOD-1 mRNA, a somatic type SOD-I mRNA (SSOD-1), is ubiquitously expressed in all somatic tissues as well as in testes. The larger SOD-1 mRNA, a testis-specific SOD-1 mRNA (TSOD-1), derived from an alternative upstream start site, is transcribed solely in postmeiotic germ cells and is translationally regulated during spermiogenesis. Since the two mRNAs have identical nucleotides except that TSOD-1 has an additional sequence at its 5' terminus, we have proposed that the extra 5' UTR sequence may be involved in the translational control of the TSOD-1 mRNA during spermiogenesis. Here we show that, when assayed in a cell-free system, TSOD-1 is translated only slightly less efficiently than SSOD-1. RNA gel retardation and UV cross-linking assays reveal that a testicular cytoplasmic protein (Cu/Zn superoxide dismutase RNA-binding protein [SOD-RBP]) of about 65 kDa specifically binds to the extended 5' UTR of TSOD-1. After purification of SOD-RBP by RNA affinity chromatography, we demonstrate that SOD-RBP can repress the in vitro translation of TSOD-1 mRNA but not SSOD-1 mRNA or cotranslated luciferase mRNA. We conclude that SOD-RBP serves as a repressor in the translation of TSOD-1 mRNA during spermiogenesis and thereby fine-tunes the level of Cu/Zn superoxide dismutase produced in maturing germ cells. PMID:8754854

  18. Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated.

    PubMed

    Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M; Olovnikov, Ivan A; Prassolov, Vladimir S; Merrick, William C; Shatsky, Ivan N

    2007-07-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

  19. Efficient Translation Initiation Directed by the 900-Nucleotide-Long and GC-Rich 5′ Untranslated Region of the Human Retrotransposon LINE-1 mRNA Is Strictly Cap Dependent Rather than Internal Ribosome Entry Site Mediated▿

    PubMed Central

    Dmitriev, Sergey E.; Andreev, Dmitri E.; Terenin, Ilya M.; Olovnikov, Ivan A.; Prassolov, Vladimir S.; Merrick, William C.; Shatsky, Ivan N.

    2007-01-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5′ untranslated region (5′UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5′UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5′UTR-Fluc) or bicistronic (Rluc-L1 5′UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5′UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5′UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5′UTR. Nevertheless, this cap-dependent initiation activity of the L1 5′UTR was unexpectedly high and resembles that of the beta-actin 5′UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5′UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5′UTRs and call into question the conception that every long GC-rich 5′UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event. PMID:17470553

  20. Tumor-promoting function of single nucleotide polymorphism rs1836724 (C3388T) alters multiple potential legitimate microRNA binding sites at the 3'-untranslated region of ErbB4 in breast cancer.

    PubMed

    Bagheri, Fatemeh; Mesrian Tanha, Hamzeh; Mojtabavi Naeini, Marjan; Ghaedi, Kamran; Azadeh, Mansoureh

    2016-05-01

    ErbB4 can act as either a tumor-suppressor gene or an oncogene in breast cancer. Multiple genetic factors including single nucleotide polymorphisms (SNPs) affect gene expression patterns. Multiple 3'-untranslated region (3'-UTR) SNPs reside within the target binding site of microRNAs, which can strengthen or weaken binding to target genes. The present study aimed to predict potential 3'‑UTR variants of ErbB4 that alter the target binding site of microRNAs (miRNAs) and to clarify the association of the potential variant with the risk of developing breast cancer. In silico prediction was performed to identify potential functional SNPs within miRNA target binding sites in the 3'‑UTR of ErbB4. Thus, 146 patients and controls were genotyped using restriction fragment length polymorphism-polymerase chain reaction. In addition to the Cochran-Armitage test for trend, allele and genotype frequency differences were determined to investigate the association between rs1836724 and the susceptibility to breast cancer. Bioinformatics analysis identified rs1836724 to be a polymorphism in the seed region of four miRNA binding sites (hsa-miR335-5p, hsa-miR-28-5p, has‑miR‑708‑5p and has‑miR‑665), which may participate in the development of breast cancer. Logistic regression data indicated that the T allele of the polymorphism [OR (95% CI)=1.72 (1.056‑2.808), P=0.029] is associated with the risk of breast cancer. Using bioinformatics tools, a correlation was indicated between the presence of the T allele and a reduction in ErbB4 RNA silencing based on miRNA interaction. Furthermore, case subgroup data analysis revealed an association between the C/T genotype and an ER positive phenotype [OR (95% CI)=6.00 (1.082‑33.274), P=0.028] compared with the T/T genotype. ErbB4 and estrogen receptor 1 (ESR1) are regulated by identical miRNAs thus there may be a competition for binding sites. Due to this pattern, if the interaction between miRNAs with one gene is reduced, it

  1. 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),…

  2. 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

  3. A novel interaction of Cap-binding protein complexes eukaryotic initiation factor (eIF) 4F and eIF(iso)4F with a region in the 3'-untranslated region of satellite tobacco necrosis virus.

    PubMed

    Gazo, Brandy M; Murphy, Patricia; Gatchel, Jennifer R; Browning, Karen S

    2004-04-01

    Satellite tobacco necrosis virus (STNV) RNA is naturally uncapped at its 5' end and lacks polyadenylation at its 3' end. Despite lacking these two hallmarks of eukaryotic mRNAs, STNV-1 RNA is translated very efficiently. A approximately 130-nucleotide translational enhancer (TED), located 3' to the termination codon, is necessary for efficient cap-independent translation of STNV-1 RNA. The STNV-1 TED RNA fragment binds to the eukaryotic cap-binding complexes, initiation factor (eIF) 4F and eIF(iso)4F, as measured by nitrocellulose binding and fluorescence titration. STNV-1 TED is a potent inhibitor of in vitro translation when added in trans. This inhibition is reversed by the addition of eIF4F or eIF(iso)4F, and the subunits of eIF4F and eIF(iso)4F cross-link to STNV-1 TED, providing additional evidence that these factors interact directly with STNV-1 TED. Deletion mutagenesis of the STNV-1 TED indicates that a minimal region of approximately 100 nucleotides is necessary to promote cap-independent translation primarily through interaction with the cap binding subunits (eIF4E or eIF(iso)4E) of eIF4F or eIF(iso)4F.

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

    PubMed Central

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

    1994-01-01

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

  5. 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.

  6. Sequence of the cDNA and 5'-flanking region for human acid alpha-glucosidase, detection of an intron in the 5' untranslated leader sequence, definition of 18-bp polymorphisms, and differences with previous cDNA and amino acid sequences.

    PubMed

    Martiniuk, F; Mehler, M; Tzall, S; Meredith, G; Hirschhorn, R

    1990-03-01

    Acid maltase or acid alpha-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose and is deficient in glycogen storage disease type II. Previously, we isolated a partial cDNA (1.9 kb) for human GAA; we have now used this cDNA to isolate and determine sequence in longer cDNAs from four additional independent cDNA libraries. Primer extension studies indicated that the mRNA extended approximately 200 bp 5' of the cDNA sequence obtained. Therefore, we isolated a genomic fragment containing 5' cDNA sequences that overlapped the previous cDNA sequence and extended an additional 24 bp to an initiation codon within a Kozak consensus sequence. The sequence of the genomic clone revealed an intron-exon junction 32 bp 5' to the ATG, indicating that the 5' leader sequence was interrupted by an intron. The remaining 186 bp of 5' untranslated sequence was identified approximately 3 kb upstream. The promoter region upstream from the start site of transcription was GC rich and contained areas of homology to Sp1 binding sites but no identifiable CAAT or TATA box. The combined data gave a nucleotide sequence of 2,856 bp for the coding region from the ATG to a stop codon, predicting a protein of 952 amino acids. The 3' untranslated region contained 555 bp with a polyadenylation signal at 3,385 bp followed by 16 bp prior to a poly(A) tail. This sequence of the GAA coding region differs from that reported by Hoefsloot et al. (1988) in three areas that change a total of 42 amino acids. Direct determination of the amino acid sequence in one of these areas confirmed the nucleotide sequence reported here but also disagreed with the directly determined amino acid sequence reported by Hoefsloot et al. (1988). At two other areas, changes in base pairs predicted new restriction sites that were identified in cDNAs from several independent libraries. The amino acid changes in all three ares increased the homology to rabbit-human isomaltase. Therefore, we believe that our

  7. 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.

  8. 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

  9. The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation.

    PubMed

    Wu, Ching-Shyi; Zou, Lee

    2016-01-01

    The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling. PMID:26565033

  10. Molecular characterization of an intragenic minisatellite (VNTR) polymorphism in the human parathyroid hormone-related peptide gene in chromosome region 12p12. 1-p11. 2

    SciTech Connect

    Pausova, Z.; Morgan, K.; Fujiwara, M.; Bourdon, J.; Goltzman, D.; Hendy, G.N. )

    1993-07-01

    The human parathyroid hormone-related peptide (hPTHrP) gene in chromosome region 12p12.1-p11.2 plays an important role in mammalian development and specifically in skeletogenesis. The authors have characterized a VNTR polymorphism in the hPTHrP gene that is located in an intron 100-bp downstream of exon VI that encodes a 3[prime] untranslated region. By PCR analysis eight different alleles were identified in a group of 112 unrelated individuals. All eight alleles were sequenced and the repeat unit was identified as the general sequence [G(TA)[sub n]C][sub N], where n = 4 to 11 and N = 3 to 17. This polymorphic sequence-tagged site will be useful for mapping chromosome 12p and will aid in testing for linkage of genetic diseases to the hPTHrP gene. 3 refs., 2 figs.

  11. 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

  12. 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.

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

    SciTech Connect

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

    1995-08-15

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

  14. 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.

  15. Characteristics of polymorphism at a VNTR locus 3[prime] to the apolipoprotein B gene in five human populations

    SciTech Connect

    Deka, R.; DeCroo, S.; Ferrell, R.E. ); Chakraborty, R.; Barton, S.A. ); Rothhammer, F. )

    1992-12-01

    The authors have analyzed the allele frequency distribution at the hypervariable locus 3[prime] to the apolipoprotein B gene (ApoB 3[prime] VNTR) in five well-defined human populations (Kacharis of northeast India, New Guinea Highlanders of Papua New Guinea, Dogrib Indians of Canada, Pehuenche Indians of Chile, and a relatively homogeneous Caucasian population of northern German extraction) by using the PCR technique. A total of 12 segregating alleles were detected in the pooled sample of 319 individuals. A fairly consistent bimodal pattern of allele frequency distribution, apparent in most of these geographically and genetically diverse populations, suggests that the ApoB 3[prime] VNTR polymorphism predates the geographic dispersal of ancestral human populations. In spite of the observed high degree of polymorphism at this locus (expected heterozygosity levels 55%-78%), the genotype distributions in all populations (irrespective of their tribal or cosmopolitan nature) conform to their respective Hardy-Weinberg predictions. Furthermore, analysis of the congruence between expected heterozygosity and the observed number of alleles reveals that, in general, the allele frequency distributions at this locus are in agreement with the predictions of the classical mutation-drift models. The data also show that alleles that are shared by all populations have the highest average frequency within populations. These findings demonstrate the potential utility of highly informative hypervariable loci such as the ApoB 3[prime] VNTR locus in population genetic research, as well as in forensic medicine and determination of biological relatedness of individuals. 38 refs., 2 figs., 3 tabs.

  16. 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

  17. 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.

  18. Do untranslated introns control Ca2+-ATPase isoform dependence on CaM, found in TN and PM?

    PubMed

    Navarro-Aviñó, Juan Pedro; Bennett, Alan B

    2003-12-26

    Transcript splicing characterization of tomato Ca(2+)-ATPase (LCA1 gene) mRNA indicates that two main transcripts are differentiated in the 3(') terminal region. One of them contains a sequence of about 90bp that could correspond to an untranslated intron that displays sequence homology to calmodulin-binding regions. Calmodulin-binding experiments demonstrate that only one of the two isoforms encoded by LCA1 binds to calmodulin. Since the M(w) calculated for this peptide is 3.7kDa, it is suggested that the presence of this intron is accounted for by the difference in the sizes of the two 116- and 120-kDa isoforms, and it determines calmodulin regulation. This represents a new strategy for a single gene to produce two isoforms that are localized differently (TN and PM), and which are either dependent on or independent of the calmodulin, which in turn is either regulated by the presence or by the absence of a 90bp untranslated intron.

  19. 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.

  20. Regional chromosomal assignments for four members of the myocyte-specific enhancer-binding factor 2 (MEF2) gene family to human chromosomes 15q, 19q, 5q, and 1q

    SciTech Connect

    Hobson, G.M.; Funanage, V.L.; Krahe, R.

    1994-09-01

    MEF2 genes belong to the MADS box family of transcription factors and encode proteins that bind as homo- and heterodimers to a consensus CTA(T/A){sub 4}TAG/A sequence present in the regulatory regions of numerous muscle-specific and growth inducible genes. Sequence analysis of human MEF2 cDNA clones suggested that they arose from alternatively spliced transcripts of four different genes, termed MEF2A-D. We have mapped the MEF2 genes to human chromosomal regions by identifying unique sequences in the 5{prime} or 3{prime} untranslated regions of each clone and using these sequences as PCR primers on the DNA of a human-rodent hybrid clone panel informative for different regions of the human genome. The localization of MEF2A to chromosome 15q, MEF2B to 19q, MEF2C to 5q, and MEF2D to 1q verifies the existence of at least four distinct loci for members of this gene family. The same PCR primers were used to identify individual YAC clones for each gene. Such isolated clones are now being used for fluorescence in situ hybridization for high resolution chromosomal regional assignment.

  1. 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).

  2. Translational control of maskin mRNA by its 3' untranslated region

    PubMed Central

    Meijer, Hedda A.; Radford, Helois E.; Wilson, Lolita S.; Lissenden, Sarah; de Moor, Cornelia H.

    2007-01-01

    Background information. Maskin is a member of the acidic transforming coiled-coil (TACC) domain proteins found in Xenopus leavis oocytes and embryos. It is implicated in the coordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. Results We report here that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3' UTR of maskin can confer the translational regulation to a reporter mRNA, and so can the 3' UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV crosslinking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3' UTRs. As previously reported, CPEB binds to the cyclin B1 3' UTR, but its binding to the maskin 3' UTR is minimal. By RNA affinity chromatography and mass spectrometry, we identified the embryonic deadenylation element binding protein (EDEN-BP) as one of the proteins binding to both the maskin and the cyclin B1 3' UTRs. Conclusion Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3' UTRs, indicating it may be involved in the deadenylation of these mRNAs. PMID:17241108

  3. The assessment of noncoding variant of PPOX gene in variegate porphyria reveals post-transcriptional role of the 5' untranslated exon 1.

    PubMed

    Fiorentino, Valeria; Brancaleoni, Valentina; Granata, Francesca; Graziadei, Giovanna; Di Pierro, Elena

    2016-10-01

    The PPOX gene encodes for the protoporphyrinogen oxidase, which is involved in heme production. The partial deficiency of protoporphyrinogen oxidase causes variegate porphyria. The tissue-specific regulation of other heme biosynthetic enzymes is extensively studied, but the information concerning transcriptional and post-transcriptional regulation of PPOX gene expression is scarcely available. In this study, we characterized functions of three variants identified in the regulatory regions of the PPOX gene, which show a novel role for the 5' untranslated exon 1. Using luciferase assays and RNA analysis, we demonstrated that only c.1-883G>C promoter variant causes a significant loss in the transcriptional activity of PPOX gene whereas c.1-413G>T 5' UTR variant inhibits translation of PPOX mRNA and c.1-176G>A splicing variant causes 4bp deletion in 5' UTR of PPOX mRNA variant 2. These observations indicate that the regulation of PPOX gene expression can also occur through a post-transcriptional modulation of the amount of gene product and that this modulation can be mediated by 5' untranslated exon 1. Moreover this study confirms that these regulatory regions represent an important molecular target for the pathogenesis of variegate porphyria. PMID:27667166

  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. 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.

  6. 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

  7. Nucleotide sequence of 3' untranslated portion of human alpha globin mRNA.

    PubMed Central

    Wilson, J T; deRiel, J K; Forget, B G; Marotta, C A; Weissman, S M

    1977-01-01

    We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A). Images PMID:909779

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

    PubMed

    Kolev, Nikolay G; Hartland, Emilia I; Huber, Paul W

    2008-10-01

    The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

  16. A novel mRNA 3' untranslated region translational control sequence regulates Xenopus Wee1 mRNA translation.

    PubMed

    Wang, Yi Ying; Charlesworth, Amanda; Byrd, Shannon M; Gregerson, Robert; MacNicol, Melanie C; MacNicol, Angus M

    2008-05-15

    Cell cycle progression during oocyte maturation requires the strict temporal regulation of maternal mRNA translation. The intrinsic basis of this temporal control has not been fully elucidated but appears to involve distinct mRNA 3' UTR regulatory elements. In this study, we identify a novel translational control sequence (TCS) that exerts repression of target mRNAs in immature oocytes of the frog, Xenopus laevis, and can direct early cytoplasmic polyadenylation and translational activation during oocyte maturation. The TCS is functionally distinct from the previously characterized Musashi/polyadenylation response element (PRE) and the cytoplasmic polyadenylation element (CPE). We report that TCS elements exert translational repression in both the Wee1 mRNA 3' UTR and the pericentriolar material-1 (Pcm-1) mRNA 3' UTR in immature oocytes. During oocyte maturation, TCS function directs the early translational activation of the Pcm-1 mRNA. By contrast, we demonstrate that CPE sequences flanking the TCS elements in the Wee1 3' UTR suppress the ability of the TCS to direct early translational activation. Our results indicate that a functional hierarchy exists between these distinct 3' UTR regulatory elements to control the timing of maternal mRNA translational activation during oocyte maturation.

  17. Breed-Dependent Transcriptional Regulation of 5′-Untranslated GR (NR3C1) Exon 1 mRNA Variants in the Liver of Newborn Piglets

    PubMed Central

    Yang, Xiaojing; Ni, Yingdong; Cong, Rihua; Soloway, Paul D.; Zhao, Ruqian

    2012-01-01

    Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P<0.05) higher in the liver of newborn piglets of Large White (LW) compared with Erhualian, a Chinese indigenous breed. Overall level of CpG methylation in the region flanking exons 1–4 and 1–5 did not show breed difference. However, nuclear content of Sp1, p-CREB and GR in the liver was significantly (P<0.05) higher in LW piglets, associated with enhanced binding of p-CREB, and higher level of histone H3 acetylation in 1–4 and 1–5 promoters. In contrast, GR binding to promoters of exons 1–4 and 1–5 was significantly diminished in LW piglets, implicating the presence of negative GREs. These results indicate that the difference in the hepatic expression of GR transcript variants between two breeds of pigs is determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3 acetylation to the promoters. PMID:22792317

  18. 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.

  19. 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

  20. 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.

  1. Degradation of 4,4{prime}-Dichlorobiphenyl, 3,3{prime}, 4,4{prime}-Tetrachlorobiphenyl, and 2,2{prime},4,4{prime},5,5{prime}-Hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium

    SciTech Connect

    Dietrich, D.; Lamar, R.; Hickey, W.J.

    1995-11-01

    The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4{prime}- dichlorobiphenyl [DCB], 3,3{prime},4,4{prime}-tetrachlorobiphenyl, and 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, {sup 14}C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the {sup 14}C was determined to be biomass bound. Results from a time course study done with 4,4{prime}-[{sup 14}C]DCB to examine {sup 14}C partitioning dynamics indicated that the biomass-bound {sup 14}C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4{prime}-[{sup 14}C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process. 23 refs., 4 figs., 2 tabs.

  2. The 5' untranslated sequence of the c-sis/platelet-derived growth factor 2 transcript is a potent translational inhibitor.

    PubMed Central

    Rao, C D; Pech, M; Robbins, K C; Aaronson, S A

    1988-01-01

    c-sis/platelet-derived growth factor 2 (PDGF-2) is a prototype growth factor with transforming potential. The c-sis/PDGF-2 transcript contains a long 5' untranslated sequence (UTS) that is highly G.C rich. To examine the influence of this sequence on sis/PDGF-2 expression, we localized the c-sis/PDGF-2 promoter and used this promoter or the simian virus 40 early promoter to drive expression of the bacterial chloramphenicol acetyltransferase or sis/PDGF-2 gene. The 5' UTS of c-sis/PDGF-2 mRNA had no effect on RNA expression but was shown to exert a potent inhibitory effect on translation. By deletion analysis, we demonstrated that the 5' UTS inhibited protein expression by as much as 40-fold. The inhibitory effect was independent of reporter gene, cell type, or promoter used. A highly G.C-rich 140-base-pair sequence immediately preceding the c-sis/PDGF-2 initiation codon was shown to be nearly as effective as the entire 5' UTS in translational inhibition. Transfection analysis demonstrated that the 5' UTS significantly reduced the transforming efficiency of the sis/PDGF-2 gene as well. Thus, our findings raise the possibility that changes in regulation at the level of sis/PDGF-2 translation may play a role in development of the neoplastic phenotype. Images PMID:3275870

  3. 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.

  4. The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency.

    PubMed Central

    Emory, S A; Belasco, J G

    1990-01-01

    The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA message can function in vivo as an mRNA stabilizer. Substitution of this ompA mRNA segment for the corresponding segment of the labile bla gene transcripts prolongs their lifetime by a factor of 6. We show here that the function of this ompA mRNA stabilizer requires the presence of a 115-nucleotide ompA RNA segment that lies upstream of the ribosome-binding site. Although deletion of this segment reduced the half-life of the ompA transcript by a factor of 5, its absence had almost no effect on the translational efficiency of ompA mRNA. Like the ompA transcript, but unlike bla mRNA, hybrid ompA-bla messages containing the complete ompA 5' UTR were significantly less stable under conditions of slow bacterial growth. We conclude that the stabilizing activity of the ompA 5' UTR is growth rate regulated and that the mechanism of mRNA stabilization by this RNA segment is not related to the spacing between translating ribosomes. Images PMID:1695894

  5. 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.

  6. Complete physical map of the human immunoglobulin heavy chain constant region gene complex

    SciTech Connect

    Hofker, M.H.; Walter, M.A.; Cox, D.W. )

    1989-07-01

    The authors have found by pulsed-field gel electrophoresis that the human immunoglobulin heavy chain constant region gene complex maps entirely to a 350-kilobase (kb) Mlu I fragment. The enzyme Eag I was used with pulsed-field gel electrophoresis alone and in double digests with Spe I to map the region. C{sub {gamma}}3 maps 60 kb to the 3{prime} side of C{sub {delta}}; C{gamma}2 maps 80 kb to the 3{prime} side of C{sub {alpha}}1. C{sub {psi}{gamma}} maps 35 kb to the 3{prime} side of C{sub {alpha}}1 and is in the same transcriptional orientation as the other genes. Although in the cloned DNA many CpG-containing restriction sites were identified, most of these were methylated in peripheral blood leukocytes. The sites that were not methylated were predominantly found in three clusters, or Hpa I tiny fragment islands. A region showing strong linkage disequilibrium between all C{sub {gamma}} genes spans at least 160 kb. The 70-kb C{sub {mu}}-C{sub {gamma}}3 region, however, shows no linkage disequilibrium, possibly indicating a recombination hot spot. The immunoglobulin heavy chain constant region has been almost entirely cloned and mapped, and thus most rearrangements occurring in this region should be detectable.

  7. Riboswitches in unexpected places--a synthetic riboswitch in a protein coding region.

    PubMed

    Topp, Shana; Gallivan, Justin P

    2008-12-01

    In natural and engineered systems, cis-RNA regulatory elements such as riboswitches are typically found within untranslated regions rather than within the protein coding sequences of genes. However, RNA sequences with important regulatory roles can exist within translated regions. Here, we present a synthetic riboswitch that is encoded within the translated region of a gene and represses Escherichia coli gene expression greater than 25-fold in the presence of a small-molecule ligand. The ability to encode riboswitches within translated regions as well as untranslated regions provides additional opportunities for creating new genetic control elements. Furthermore, evidence that a riboswitch can function in the translated region of a gene suggests that future efforts to identify natural riboswitches should consider this possibility. PMID:18945803

  8. Assessment of microRNA-related SNP effects in the 3' untranslated region of the IL22RA2 risk locus in multiple sclerosis.

    PubMed

    Lill, Christina M; Schilling, Marcel; Ansaloni, Sara; Schröder, Julia; Jaedicke, Marian; Luessi, Felix; Schjeide, Brit-Maren M; Mashychev, Andriy; Graetz, Christiane; Akkad, Denis A; Gerdes, Lisa-Ann; Kroner, Antje; Blaschke, Paul; Hoffjan, Sabine; Winkelmann, Alexander; Dörner, Thomas; Rieckmann, Peter; Steinhagen-Thiessen, Elisabeth; Lindenberger, Ulman; Chan, Andrew; Hartung, Hans-Peter; Aktas, Orhan; Lohse, Peter; Buttmann, Mathias; Kümpfel, Tania; Kubisch, Christian; Zettl, Uwe K; Epplen, Joerg T; Zipp, Frauke; Bertram, Lars

    2014-05-01

    Recent large-scale association studies have identified over 100 MS risk loci. One of these MS risk variants is single-nucleotide polymorphism (SNP) rs17066096, located ~14 kb downstream of IL22RA2. IL22RA2 represents a compelling MS candidate gene due to the role of IL-22 in autoimmunity; however, rs17066096 does not map into any known functional element. We assessed whether rs17066096 or a nearby proxy SNP may exert pathogenic effects by affecting microRNA-to-mRNA binding and thus IL22RA2 expression using comprehensive in silico predictions, in vitro reporter assays, and genotyping experiments in 6,722 individuals. In silico screening identified two predicted microRNA binding sites in the 3'UTR of IL22RA2 (for hsa-miR-2278 and hsa-miR-411-5p) encompassing a SNP (rs28366) in moderate linkage disequilibrium with rs17066096 (r (2) = 0.4). The binding of both microRNAs to the IL22RA2 3'UTR was confirmed in vitro, but their binding affinities were not significantly affected by rs28366. Association analyses revealed significant association of rs17066096 and MS risk in our independent German dataset (odds ratio  = 1.15, P = 3.48 × 10(-4)), but did not indicate rs28366 to be the cause of this signal. While our study provides independent validation of the association between rs17066096 and MS risk, this signal does not appear to be caused by sequence variants affecting microRNA function.

  9. Pilot study of the reducing effect on amyloidosis in vivo by three FDA pre-approved drugs via the Alzheimer's APP 5' untranslated region.

    PubMed

    Tucker, Stephanie; Ahl, Michelle; Bush, Ashley; Westaway, David; Huang, Xudong; Rogers, Jack T

    2005-04-01

    A pilot study was conducted employing a well known mouse model for Alzheimer's disease to evaluate the anti-amyloid efficacy of three FDA pre-approved drugs. Paroxetine (SSRI and APP 5'UTR directed lead compound), N-acetyl cysteine (antioxidant), and erythromycin (macrolide antibiotic) were provided to the drinking water of TgCRND8 mice for three months. This report provides data that measured the steady-state levels of amyloid Abeta-40 and Abeta-42 Abeta as pmol Abeta per gram of mouse brain cortex in drug treated and placebo animals. The relative levels of Abeta peptide levels were reduced after exposure of mice to paroxetine (N=5), NAC (N=7), and erythromycin (N=7) relative to matched placebo counterparts. These results demonstrated proof-of concept for a strategy to further screen the APP 5'UTR target to identify novel drugs that exhibit anti-amyloid efficacy in vivo. These data also demonstrated a statistically significant anti-amyloid trend for paroxetine, NAC and erythromycin. The potential for conducting further studies with these compounds using larger cohorts of TgCRND8 mice is discussed.

  10. Eukaryotic initiation factor (eIF) 4F binding to barley yellow dwarf virus (BYDV) 3'-untranslated region correlates with translation efficiency.

    PubMed

    Banerjee, Bidisha; Goss, Dixie J

    2014-02-14

    Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3'-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the "on" rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52-90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3' BTE with higher affinity than for either m(7)G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3' BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation. PMID:24379412

  11. 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.

  12. Truncated form of VACM-1/cul-5 with an extended 3' untranslated region stimulates cell growth via a MAPK-dependent pathway

    SciTech Connect

    Sartor, Ashleigh; Kossoris, J.B.; Wilcox, R.; Shearer, R.; Zeneberg, A.E.; Zhao, P.; Lazdins, I.; Burnatowska-Hledin, Maria A. . E-mail: hledin@hope.edu

    2006-05-19

    We have sequenced a 4.9 kb clone (KLB22) which shares 99% sequence homology with the rabbit vasopressin-activated calcium mobilizing (VACM-1) protein. The 5' terminus sequence of KLB22 cDNA (nucleotides 1-1961) is continuous and overlapping with nucleotides 1226-3186 of the VACM-1 cDNA sequence. The 3'UTR of KLB22 cDNA extends beyond the 3'UTR of VACM-1 by 2999 nt. KLB22 cDNA encodes a 497 amino acid protein, which putatively begins at Met 284 of the 780 amino acid VACM-1 protein. The in vitro translation of KLB22 cDNA yields a 59 kDa protein. When expressed in cos-1 cells, the truncated VACM-1 protein localizes to the nucleus. KLB22 cDNA transfected cells show increased growth rates and increased levels of phosphorylated MAPK when compared to the vector or to VACM-1 cDNA transfected cells. Finally, in vivo, KLB22 protein expression is tissue specific and can be detected in kidney and in heart atrium. These results suggest that truncated VACM-1 cDNA (KLB22) increases cell proliferation through a MAPK pathway.

  13. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. The C421A (Q141K) polymorphism enhances the 3'-untranslated region (3'-UTR)-dependent regulation of ATP-binding cassette transporter ABCG2.

    PubMed

    Ripperger, Anne; Benndorf, Ralf A

    2016-03-15

    The impact of the gout-causing C421A (Q141K) single nucleotide polymorphism (SNP) on ABC transporter ABCG2 expression and function has been extensively characterized. However, the influence of the C421A SNP on 3'-UTR-dependent ABCG2 regulation has not been analysed so far. To elucidate this matter, we generated vectors for expression of either the ABCG2 coding sequence (ORF) or the ABCG2 ORF fused to its 3'-UTR, inserted the C421A mutation via site-directed mutagenesis and expressed wild-type and C421A-mutated ABCG2 transcripts in HEK293-Tet-On cells. As shown previously, the C421A SNP significantly reduced ABCG2 protein levels in ABCG2 ORF-transfected HEK293-Tet-On cells. Interestingly, the presence of the 3'-UTR in the ABCG2 transcript dramatically reduced ABCG2 protein content in cells transfected with the C421A variant but not significantly in those transfected with ABCG2 wild-type sequence, whereas ABCG2 mRNA levels were similar. siRNA-mediated DICER1 knockdown to reduce cellular microRNA biogenesis and selective mutation of putative microRNA binding sites within the ABCG2 3'-UTR partially antagonized C421A-associated reduction of ABCG2 protein content but did not significantly affect wild-type ABCG2 protein levels. In addition, antagomir-mediated inhibition of two microRNAs (hsa-miR-519c and hsa-miR-328) again partially reversed C421A-associated ABCG2 translational repression, thereby indicating that the C421A SNP may facilitate microRNA-dependent repression of ABCG2 protein translation. We conclude from our results that the C421A SNP may lead to reduced ABCG2 protein levels not only by affecting cellular protein stability but also via enhanced microRNA-dependent ABCG2 repression. Moreover, tissue-specific variation in ABCG2 3'-UTR processing may profoundly affect ABCG2 expression levels in individuals carrying the C421A mutation. PMID:26903388

  16. Prohibitin Expression Deregulation in Gastric Cancer Is Associated with the 3′ Untranslated Region 1630 C>T Polymorphism and Copy Number Variation

    PubMed Central

    Leal, Mariana Ferreira; Cirilo, Priscila Daniele Ramos; Mazzotti, Tatiane Katsue Furuya; Calcagno, Danielle Queiroz; Wisnieski, Fernanda; Demachki, Samia; Martinez, Margarita Cortes; Assumpção, Paulo Pimentel; Chammas, Roger; Burbano, Rommel Rodríguez; Smith, Marília Cardoso

    2014-01-01

    PHB is a reported oncogene and tumor suppressor in gastric cancer. Here, we evaluated whether the PHB copy number and the rs6917 polymorphism affect its expression in gastric cancer. Down-regulation and up-regulation of PHB were observed in the evaluated tumors. Reduced expression was associated with tumor dedifferentiation and cancer initiation. The T allele of the rs6917 polymorphism was associated with reduced PHB mRNA levels. Moreover, the up-regulation of PHB appeared to be regulated by the gain of additional gene copies. Thus, PHB copy number variation and differential expression of the rs6917 polymorphism may play a role in PHB transcriptional regulation. PMID:24879411

  17. Prohibitin expression deregulation in gastric cancer is associated with the 3' untranslated region 1630 C>T polymorphism and copy number variation.

    PubMed

    Leal, Mariana Ferreira; Cirilo, Priscila Daniele Ramos; Mazzotti, Tatiane Katsue Furuya; Calcagno, Danielle Queiroz; Wisnieski, Fernanda; Demachki, Samia; Martinez, Margarita Cortes; Assumpção, Paulo Pimentel; Chammas, Roger; Burbano, Rommel Rodríguez; Smith, Marília Cardoso

    2014-01-01

    PHB is a reported oncogene and tumor suppressor in gastric cancer. Here, we evaluated whether the PHB copy number and the rs6917 polymorphism affect its expression in gastric cancer. Down-regulation and up-regulation of PHB were observed in the evaluated tumors. Reduced expression was associated with tumor dedifferentiation and cancer initiation. The T allele of the rs6917 polymorphism was associated with reduced PHB mRNA levels. Moreover, the up-regulation of PHB appeared to be regulated by the gain of additional gene copies. Thus, PHB copy number variation and differential expression of the rs6917 polymorphism may play a role in PHB transcriptional regulation. PMID:24879411

  18. A stem–loop structure in the 59 untranslated region of bean pod mottle virus RNA2 is specifically required for RNA2 accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  19. Variation in the coding and 3’ untranslated regions of the porcine prolactin receptor short form modifies protein expression and function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. The short 5' untranslated region of the betaA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning.

    PubMed

    Werten, P J; Stege, G J; de Jong, W W

    1999-08-01

    Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. BetaA3- and betaA1-crystallin, two members of the beta-crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the betaA3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of betaA1-crystallin. It has been suggested that the very short leader (5-7 nucleotides) of the betaA3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the betaA1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both betaA3- and betaA1-crystallin at very similar relative levels.

  1. 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.

  2. CERKL, a Retinal Disease Gene, Encodes an mRNA-Binding Protein That Localizes in Compact and Untranslated mRNPs Associated with Microtubules

    PubMed Central

    Riera, Marina; Knecht, Erwin; Gonzàlez-Duarte, Roser

    2014-01-01

    The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases. PMID:24498393

  3. 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.

  4. In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities

    SciTech Connect

    Sera, T.; Schultz, P.G.

    1996-04-02

    A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn{sup {minus}18}, Ala{sup {minus}15}, Val{sup {minus}14}, Ser{sup {minus}11}, And Arg{sup {minus}10}. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5{prime}-A{sup 5}T{sup 4}T{sup 3}G{sup 2}C{sup 1}G{sup 1{prime}}C{sup 2{prime}}A{sup 3}A{sup 4{prime}}T{sup 5{prime}}-3{prime}. Mutants containing the sequence Leu{sup {minus}18}Tyr{sup {minus}15}Xaa{sup {minus}14}-Tyr{sup {minus}11}Arg{sup {minus}10}, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5{prime}-ACTCYCGY{prime}GAT-3{prime} (Xaa=asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5{prime}-ATTACGTAAT-3{prime}, mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed. 28 refs., 1 fig., 4 tabs.

  5. 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.

  6. Synthesis and characterization of N,N{prime},N{double_prime},N{double_prime}{prime}-tetrasalicylidene-3,3{prime}-diaminobenzidine Schiff-base coordination polyelectrolytes of yttrium(III), lanthanum(III), gadolinium(III), and ytterbium(III)

    SciTech Connect

    Chen, H.; Archer, R.D.

    1995-02-27

    A series of linear coordination polyelectrolytes formed by lanthanide(III) ions (Ln{sup 3+} = La{sup 3+}, Gd{sup 3+}, Y{sup 3+}, or Yb{sup 3+}) with the anion of the Schiff-base N,N{prime},N{double_prime}{prime}-tetrasalicylidene-3,3{prime}-diaminobenzidine (H{sub 4}tsdb) have been synthesized. This new series has the formula [MLn(tsdb)]{sub n} (M{sup +} = an alkali metal ion). Some can dissolve in polar organic solvents with high dielectric constants, such as DMSO, NMP, and DMF, and they exhibit the typical properties of polyelectrolytes, i.e., an apparent increase in inherent viscosity for dilutes solutions; the formation of colloidal precipitates when water is added to the solutions; smaller solution conductivity relative to simple electrolytes; high thermal stability (>773 K); and high glass transition temperature. Molecular weights of [NaY(tsdb)]{sub n} have been estimated by NMR end-group analysis to be as high as 18,500. The Mark-Houwink constants for [NaY(tsdb)]{sub n} are a = 0.510 and K = 8.12 {times} 10{sup {minus}2} cm{sup 3}/g. The molecular weights of the other polyelectrolytes have been estimated from their intrinsic viscosities. The solubilities of the polyelectrolytes are strongly affected by the counterions. Only the sodium salts of the polyelectrolytes are soluble in polar organic solvents, except for La(III), for which the lithium salt also has some solubility. The La(III) compounds give a behavior somewhat different from that of the other polyelectrolytes.

  7. Trans-4,4{prime}-dichloro-1,1{prime},2,2{prime},3,3{prime}-tetrathiadiazafulvalene (DC-TAF) and its 1:1 radical cation salts [DC-TAF][X]: Preparation and solid-state properties of BF{sub 4{minus}}, ClO{sub 4{minus}}, and FSO{sub 3{minus}} derivatives

    SciTech Connect

    Barclay, T.J.; Beer, L.; Cordes, A.W.; Haddon, R.C.; Itkis, M.I.; Oakley, R.T.; Preuss, K.E.; Reed, R.W.

    1999-07-21

    Reductive coupling of 4,5-dichloro-1,2,3-dithiazolylium chloride yields trans-4,4{prime}-dichloro-1,1{prime},2,2{prime},3,3{prime}-tetrathiadiazafulvalene (DC-TAF), the first example of this heterofulvalene system. Ab initio molecular orbital (B3LYP/6-31G**) calculations on prototypal TAF confirm that the closed shell {sup 1}A{sub g} state lies 22 kcal mol{sup {minus}1} below the {sup 3}B{sub u} diradical triplet. Cyclic voltammetry on DC-TAF reveals two reversible oxidation waves at 0.80and 1.25 V (in CH{sub 3}CN, reference SCE). The EST signal (g = 2.0117) of the radical cation [DC-TAF]{sup +} (in SO{sub 2}(1)) exhibits a five=line hyperfine coupling pattern with a{sub n} = 0.096 mT. DC-TAF forms a series of 1:1 radical ion salts [DC-TAF][X] by electrooxidation in the presence of tetrahedral counterions (X{sup {minus}} = BF{sub 4}{sup {minus}}, ClO{sub 4}{sup {minus}}, FSO{sub 3}{sup {minus}}). The crystal structures of these salts are isomorphous, monoclinic space group P2{sub 1}/n. and consist of one-dimensional ladder-like arrays of [DC-TAF]{sup +} radical cations bridged by S---S contacts ranging from 3.5 to 3.7 {angstrom}. Variable-temperature conductivity and magnetic measurements on [DC-TAF][ClO{sub 4}] indicate Mott insulator behavior, with a measured band gap of 0.30 eV.

  8. Isolation and characterization of human cDNA clones encoding the. alpha. and the. alpha. prime subunits of casein kinase II

    SciTech Connect

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs E.G. )

    1990-09-11

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two {alpha} or {alpha}{prime} subunits (or one of each) and two {beta} subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell {lambda}gt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5{prime} untranslated region) and followed by 871 bp (3{prime} untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5{prime} untranslated region) and followed by 550 bp (3{prime} untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of {alpha} and {alpha}{prime} subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the {alpha} and {alpha}{prime} subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II ({alpha} and {alpha}{prime}) and that the sequence of these subunits is largely conserved between the bovine and the human.

  9. The structural organization of the human Na{sup +}/Myo-inositol cotransporter (SLC5A3) gene and characterization of the promoter

    SciTech Connect

    Mallee, J.J.; Lucente, A.D.; Wang, Yi ||

    1997-12-15

    The genomic structure, transcription start site, polyadenylation signals, and promoter of the human Na{sup +}/myo-inositol cotransporter (SLC5A3) gene have been elucidated through cloning, sequencing, mRNA analyses, and reporter gene assays. The gene consists of one promoter and two exons spanning approximately 26 kb. Exon 1 contains 175 bp of 5{prime} untranslated sequence and is 15 kb upstream of exon 2. The 9.5-kb exon 2 contains the entire 2157-bp open reading frame and a large 3{prime} untranslated sequence with seven putative polyadenylation signals. Multiple messages with different-sized 3{prime} untranslated regions can be detected on Northern blots. Hypertonic stress caused mRNA levels, and primarily that of the full-length 9.5-kb transcript, to increase in cultured melanoma cells; ribonuclease protection analysis demonstrated that the transcription start site was the same in stressed as in control cells. The SLC5A3 gene functions in cellular osmoregulation and is expressed in many human tissues including the brain, kidney, and placenta. It is localized to chromosome 21q22.1. An overexpression of the SLC5A3 gene deserves consideration as a factor in the pathophysiology of Down syndrome. 36 refs., 4 figs., 1 tab.

  10. 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

  11. Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA, via the eIF4 Complex, Facilitates Cap-independent Translation*

    PubMed Central

    Sharma, Sohani Das; Kraft, Jelena J.; Miller, W. Allen; Goss, Dixie J.

    2015-01-01

    Barley yellow dwarf virus mRNA, which lacks both cap and poly(A) tail, has a translation element (3′-BTE) in its 3′-UTR essential for efficient translation initiation at the 5′-proximal AUG. This mechanism requires eukaryotic initiation factor 4G (eIF4G), subunit of heterodimer eIF4F (plant eIF4F lacks eIF4A), and 3′-BTE-5′-UTR interaction. Using fluorescence anisotropy, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) analysis, and toeprinting, we found that (i) 40S subunits bind to BTE (Kd = 350 ± 30 nm), (ii) the helicase complex eIF4F-eIF4A-eIF4B-ATP increases 40S subunit binding (Kd = 120 ± 10 nm) to the conserved stem-loop I of the 3′-BTE by exposing more unpaired bases, and (iii) long distance base pairing transfers this complex to the 5′-end of the mRNA, where translation initiates. Although 3′-5′ interactions have been recognized as important in mRNA translation, barley yellow dwarf virus employs a novel mechanism utilizing the 3′-UTR as the primary site of ribosome recruitment. PMID:25792742

  12. Human dopamine transporter gene (DAT1) maps to chromosome 5p15. 3 and displays a VNTR

    SciTech Connect

    Vandenbergh, D.J.; Perisco, A.M.; Uhl, G.R.; Hawkins, A.L.; Griffin, C.A.; Li, Xiang; Jabs, E.W. )

    1992-12-01

    The human dopamine transporter (DAT1) gene is localized to chromosome 5p15.3 by in situ hybridization and PCR amplification of rodent somatic cell hybrid DNA. Analysis of a 40-bp repeat in the 3[prime] untranslated region of the message revealed variable numbers of the repeat ranging from 3 to 11 copies. These results will aid in the investigation of a role for this gene in genetic disorders of the dopaminergic system in humans. 15 refs., 1 fig., 1 tab.

  13. 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.

  14. Conserved sequence elements in the 5' region of the Ultrabithorax transcription unit

    PubMed Central

    Wilde, C. Deborah; Akam, Michael

    1987-01-01

    Clones homologous to the 5' region of the Ultrabithorax gene of Drosophila melanogaster have been isolated from D. pseudoobscura, D. funebris and Musca domestica. Regions that encode most of the Ubx protein have been sequenced in all three of these species, and the 5' upstream region has been sequenced in D. funebris to a point ˜1000 bases upstream of the probable mRNA start site. Here we compare these sequences with those described elsewhere for D. melanogaster. Deduced amino acid sequences of the Ubx protein show 8% (D. pseudoobscura), 15% (D. funebris) and 22% (M. domestica) divergence from D. melanogaster. However, these figures mask very different rates of evolution in different regions of the protein. A glycine-rich (`hinge') region is conserved in each of these species, although its length is variable. Comparison of D. funebris and D. melanogaster sequences in the long 5' untranslated leader region of the mRNA, and in the region immediately upstream of the start point of transcription, reveals tightly conserved elements embedded in an otherwise non-homologous sequence. These conserved elements include a 118-bp region that spans the mRNA start site, an internally repetitive (TAA)n region in the untranslated leader and a short repeated motif immediately upstream of the ATG codon that initiates the major open reading frame of the Ubx protein. Two other conserved elements were identified upstream of the transcription start site; both elements have structural features consistent with a role as recognition sites for regulatory proteins. ImagesFig. 2. PMID:16453766

  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. Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.

    PubMed

    Berdoz, J; Monath, T P; Kraehenbuhl, J P

    1995-04-01

    We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa/lambda sequences. Variable regions with intact coding sequences, including full-length leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments that do not need to be adapted for recombinant antibody expression, thus facilitating the generation of chimeric and isotype-switched immunoglobulins.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. The regulatory region of the human plasminogen activator inhibitor type-1 (PAI-1) gene.

    PubMed Central

    Riccio, A; Lund, L R; Sartorio, R; Lania, A; Andreasen, P A; Danø, K; Blasi, F

    1988-01-01

    The human gene for plasminogen activator inhibitor type-1 (PAI-1) has been isolated and its promoter region characterized. PAI-1 regulation by glucocorticoids, transforming growth factor-beta (TGF-beta) and the phorbol ester PMA is shown to be exerted at the promoter level. A fragment spanning 805 nucleotides of the 5' flanking and 72 of the 5' untranslated region contain information enough to promote transcription and to respond to glucocorticoids when fused to a reporter gene and transfected into human fibrosarcoma cells. A moderately repetitive DNA sequence, containing a TATA box, a GRE consensus, a Z-DNA forming sequence and two imperfect direct repeats at the extremities, is present a few nucleotides 5' of the human PAI-1 gene transcription start site, raising the possibility that this gene could have been activated by DNA insertion during evolution. Images PMID:3130610

  2. Severe Gardner syndrome in families with mutations restricted to a specific region of the APC gene

    SciTech Connect

    Davies, D.R.; Armstrong, J.G.; Thakker, N.

    1995-11-01

    Familial adenomatous polyposis (FAP) is associated with a number of extraintestinal manifestations, which include osteomas, epidermoid cysts, and desmoid tumors, often referred to as {open_quotes}Gardner syndrome.{close_quotes} Recent studies have suggested that some of the phenotypic features of FAP are dependent on the position of the mutation within the APC gene. In particular, the correlation between congenital hypertrophy of the retinal pigment epithelium (CHRPE) and APC genotype indicates that affected families may be divided into distinct groups. We have investigated the association between the dento-osseous features of GS on dental panoramic radiographs (DPRs) and APC genotype in a regional cohort of FAP families. DPRs were performed on 84 affected individuals from 36 families, and the dento-osseous features of FAP were quantified by a weighted scoring system. Significant DPR abnormalities were present in 69% of affected individuals. The APC gene mutation was identified in 27 of these families, and for statistical analysis these were subdivided into three groups. Group 1 comprised 18 affected individuals from seven families with mutations 5{prime} of exon 9; these families (except one) did not express CHRPE. Groups 2 comprised 38 individuals from 16 families with mutations between exon 9 and codon 1444, all of whom expressed CHRPE. Group 3 comprised 11 individuals from four families with mutations 3{prime} of codon 1444, none of whom expressed CHRPE. Families with mutations 3{prime} of codon 1444 had significantly more lesions on DPRs (P < .001) and appeared to have a higher incidence of desmoid tumors. These results suggest that severity of some of the features of Gardner syndrome may correlate with genotype in FAP. 32 refs., 2 figs., 2 tabs.

  3. 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.

  4. 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.

  5. Isolation and characterization of the human parathyroid hormone-like peptide gene

    SciTech Connect

    Mangin, M.; Ikeda, K.; Dreyer, B.E.; Broadus, A.E. )

    1989-04-01

    A parathyroid hormone-like peptide (PTH-LP) has recently been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The peptide appears to be encoded by a single-copy gene that gives rise to multiple mRNAs that are heterogeneous at both their 5{prime} and their 3{prime} ends. Alternative RNA splicing is responsible for the 3{prime} heterogeneity and results in mRNAs encoding three different peptides, each with a unique C terminus. The authors have isolated and characterized the human PTHLP gene. The gene is a complex transcriptional unit spanning more than 12 kilobases of DNA and containing six exons. Two 5{prime} exons encode distinct 5{prime} untranslated regions and are separated by a putative promoter element, indicating that the gene either has two promoters or is alternatively spliced from a single promoter upstream of the first exon. The middle portion of the PTHLP gene, comprising exons 2-4, has an organizational pattern of introns and exons identical to that of the parathyroid hormone gene, consistent with a common ancestral origin of these two genes. Exon 4 of the PTHLP gene encodes the region common to all three peptides and the C terminus of the shortest peptide, and exons 5 and 6 encode the unique C termini of the other two peptides. Northern analysis of mRNAs from four human tumors of different histological types reveals the preferential use of 3{prime} splicing patterns of individual tumors.

  6. Sequence of the 3'-noncoding and adjacent coding regions of human gamma-globin mRNA.

    PubMed Central

    Poon, R; Kan, Y W; Boyer, H W

    1978-01-01

    In cloning human fetal globin cDNA in bacterial plasmids, we obtained a recombinant which contained a fragment of gammg-globin cDNA corresponding to the region from amino acid 99 to the poly A. We determined a sequence of 169 nucleotides which included the complete 3' non-coding region of the gamma-globin mRNA. The codon for amino acid 136 was GCA, indicating that this cloned fragment was derived from the Agamma-globin gene. In conjunction with the surrounding sequences, the GCA codon provides the Agamma-species with a unique CTGCAG hexanucleotide that is recognized by the restriction enzyme Pst I. The 3'-untranslated region of the gamma-globin mRNA consists of 90 nucleotides, and shares little homology with that of the human beta-globin mRNA. As in other mammalian mRNAs, a symmetrical sequence and the hexanucleotide AAUAAA are present. Images PMID:318163

  7. Regional odontodysplasia.

    PubMed

    Mehta, D N; Bailoor, D; Patel, B

    2011-01-01

    Regional odontodysplasia is an unusual developmental anomaly in which ectodermal and mesodermal tooth components are affected. We present a rare case of a developmental anomaly called regional odontodysplasia or 'ghost teeth' in a 12-year-old Indian girl. The anomaly affected right maxillary permanent teeth. The mandibular teeth were unaffected. The clinical, radiographic and histological features are reviewed. The management of affected patients is discussed.

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

    SciTech Connect

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

    1993-01-01

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

  9. 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.

  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. 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.

  12. Nucleotide sequence of the hypervariable region of the human C2 gene

    SciTech Connect

    Zhu, Z.B.; Volanakis, J.V. )

    1991-03-15

    It has been previously suggested that the multiallelic Bam H1/Sst I RFLPs of the human C2 gene arose through deletion/insertion of a tandemly-repeated minisatellite region. In this study the authors subcloned and sequenced the Sst I polymorphic fragment of the b haplotype of the C2 gene. This restriction fragment is 2,450 bp long and maps 1,550 bp 3{prime} of exon 3. Its nucleotide sequence is characterized by the presence of at least 4 different repeated regions varying in size from 18 to 58 bp. One of these regions starting at position 1,413 is 48 bp long and is repeated five times. The first 3 repeats are in tandem and are separated by 72 bp from two additional tandem repeats. Sequence homology among the 5 repeats ranges between 93 and 98%. Eighty three percent of the nucleotides of the repeated-region are G or C. It seems likely that this nucleotide repeat resulted in the multiallelic RFLPs through a mechanism of unequal recombination or replication slippage.

  13. Evidence for Widespread Positive and Negative Selection in Coding and Conserved Noncoding Regions of Capsella grandiflora

    PubMed Central

    Platts, Adrian E.; Hazzouri, Khaled M.; Haudry, Annabelle; Blanchette, Mathieu; Wright, Stephen I.

    2014-01-01

    The extent that both positive and negative selection vary across different portions of plant genomes remains poorly understood. Here, we sequence whole genomes of 13 Capsella grandiflora individuals and quantify the amount of selection across the genome. Using an estimate of the distribution of fitness effects, we show that selection is strong in coding regions, but weak in most noncoding regions, with the exception of 5′ and 3′ untranslated regions (UTRs). However, estimates of selection on noncoding regions conserved across the Brassicaceae family show strong signals of selection. Additionally, we see reductions in neutral diversity around functional substitutions in both coding and conserved noncoding regions, indicating recent selective sweeps at these sites. Finally, using expression data from leaf tissue we show that genes that are more highly expressed experience stronger negative selection but comparable levels of positive selection to lowly expressed genes. Overall, we observe widespread positive and negative selection in coding and regulatory regions, but our results also suggest that both positive and negative selection on plant noncoding sequence are considerably rarer than in animal genomes. PMID:25255320

  14. Gene organization of the pregnancy-specific glycoprotein region on human chromosome 19: Assembly and analysis of a 700-kb cosmid contig spanning the region

    SciTech Connect

    Olsen, A.; Nelson, D.; Gordon, L.

    1994-10-01

    The pregnancy-specific glycoprotein (PSG) gene family consists of 11 closely related genes that form a subgroup of the carcinoembryonic antigen (CEA) gene family on 19q13.2. Using a high-resolution restriction fragment fingerprinting technique, we have assembled 256 cosmids from the PSG region into a single 700-kb contig. Fluorescence in situ hybridization to sperm pronuclei and cosmid walking experiments indicated that this PSG contig was directly telomeric of CGM8 at the telomeric end of the CEA subgroup gene cluster. Detailed restriction mapping and hybridization with gene-specific probes indicated that the order of the 11 previously identified PSG genes is cen - PSG3 - PSG8 - PSG12 - PSG1 - PSG6 - PSG7 - PSG13 - PSG2 - PSG5 - PSG4 - PSG11 - tel. The CEA subgroup gene CGM11 is located at the telomeric end of the PSG gene cluster. The PSG gene are all oriented in tandem with the 5{prime}-3{prime} direction of transcription from telomere to centromere. The detailed map also led to the identification of seven new CEA family genes in the region. One of these (CGM12), located between CGM8 and PSG3, is a member of the CEA subgroup. The remaining six (CGM13 through CGM18) are interspersed among the PSG genes and appear to form a third distinct subgroup within the CEA gene family. 34 refs., 6 figs.

  15. Characterization of the gene causing type 1 spinocerebellar ataxia and identification of the murine homolog

    SciTech Connect

    Banfi, S.; Servadio, A.; McCall, A.E.

    1994-09-01

    Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant disorder characterized by neurodegeneration of the cerebellum and brainstem. The mutation in SCA1 involves the expansion of a highly polymorphic CAG trinucleotide repeat located within the coding region of a novel gene on the short arm of human chromosome 6. The SCA1 transcript is 10,660 bases and has a wide pattern of expression. The gene product, ataxin-1, is predicted to contain 792-825 amino acids (depending on the size of the CAG repeat on normal alleles) and does not share any homology with any known protein. The structure of this gene is unusual in that it contains seven exons in the 5{prime} untranslated region (5{prime} UTR) and two large exons (2080 and 7805 bp respectively) which contain the coding region, and a 7277 bp 3{prime} untranslated region (3{prime} UTR). In order to identify putative functional domains of ataxin-1 and to investigate the significance of the long 5{prime} UTR, we began characterizing the murine homolog of the SCA1 gene (Sca1). Northern analysis revealed that the size of the Sca1 transcript is approximately 10.5 kb. Sequence analysis of more than 3 kb of the murine gene revealed that Sca1 encodes for a predicted protein of 792 amino acids which shows 89% peptide identity with the human protein. The murine Sca1 gene contains only two CAG repeats suggesting that the polyglutamine tract is not essential for the normal function of this protein. Preliminary analysis of the murine locus suggests that it is very similar to the human locus with two large exons containing the coding region and a very long 3{prime} UTR. Sequence homology between the mouse and human homologs extends into the 5{prime} UTR and 3{prime} UTR with 85% and 63% identity respectively. Detailed characterization of the 5{prime} UTR in the mouse is currently in progress to determine its potential role in the regulation of transcription and/or translation of this gene.

  16. 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.

  17. 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

  18. 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.

  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. Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter.

    PubMed

    Man, Michal; Epel, Bernard L

    2004-06-01

    A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3'-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3' terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem-loop structures, which are followed by an enhancer region.

  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. 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.

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

    SciTech Connect

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

    1996-03-01

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

  4. Interferon. gamma. response region in the promoter of the human DPA gene

    SciTech Connect

    Yang, Zhi; Sugawara, Minoru; Ponath, P.D.; Wessendorf, L.; Banerji, J.; Li, Yi; Strominger, J.L. )

    1990-12-01

    The interferon {gamma} (IFN-{gamma}) response region of the human class II major histocompatibility complex gene, DPA, has been localized to a 52-base-pair (bp) DNA fragment in the proximal promotor at {minus}107 to {minus}55 bp after transfection into HeLa cells of a series of 5{prime}, 3{prime}, and gap deletion mutants linked to a reporter gene, human growth hormone, as well as of synthetic oligonucleotides fused to the heterologous promoter thymidine kinase. The 52-mer sequence contains the X and Y box elements conserved in all class II genes; their presence is indispensable for IFN-{gamma} inducibility. Furthermore, an additional 5 bp immediately 5{prime} of the X box of the DPA gene are necessary and sufficient for IFN-{gamma} induction. This region may contain an IFN-{gamma} response element. A closely related sequence has also been found in the vicinity of the critical deletion sites of three other well-studied class II gene promoters, all of which require a much longer sequence 5{prime} of the X box. A fourth element, the W element, located about 15 bp 5{prime} of the X box in all class II genes, is clearly of little importance in IFN-{gamma} inducibility of the DPA gene.

  5. 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.

  6. 5' control regions of the apolipoprotein(a) gene and members of the related plasminogen gene family.

    PubMed Central

    Wade, D P; Clarke, J G; Lindahl, G E; Liu, A C; Zysow, B R; Meer, K; Schwartz, K; Lawn, R M

    1993-01-01

    Elevated blood levels of apolipoprotein(a), the component of lipoprotein(a) that distinguishes it from low density lipoprotein, are a major risk factor for atherosclerosis. The apolipoprotein(a) gene is highly similar to the plasminogen gene and to at least four other genes or pseudogenes. The 5' untranslated and flanking sequences of these six genes contain extensive regions of near identity and share sequence elements involved in the initiation of transcription and translation. About 1000 base pairs of flanking DNA of each gene are sufficient to promote transcription in cultured hepatocytes. The apolipoprotein(a) gene promoter contains functional interleukin 6-responsive elements, consistent with the reported acute-phase response of apolipoprotein(a). Flanking genomic fragments of the apoliprotein(a) gene from two individuals with vastly different plasma apolipoprotein(a) concentrations have sequence differences that are reflected in differences in the rate of in vitro transcription. Images PMID:7679504

  7. 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.

  8. 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.

  9. Characterization and polymerase chain reaction (PCR) detection of an Alu deletion polymorphism in total linkage disequilibrium with myotonic dystrophy

    SciTech Connect

    Mahadevan, M.S. ); Foitzik, M.A. ); Surh, L.C.; Korneluk, R.G. Univ. of Ottawa )

    1993-02-01

    The mutation causing myotonic dystrophy has been identified as an unstable trinucleotide CRG repeat located in the 3[prime] untranslated region of a gene putatively encoding a serine-threonine protein kinase. The mutation has been reported to be in total linkage disequilibrium with an insertion/deletion polymorphism located within the kinase gene. To determine the nature of this polymorphism, we have sequenced this genomic fragment and have found that the sequence of this region consists of five consecutive Alu repeats. Further analysis suggests that the smaller of two alleles is actually due to a proposed deletion event that resulted in the loss of an equivalent of three Alu repeats. We have developed a PCR-based assay to detect this polymorphism, the closest, distal marker to the DM mutation. 12 refs., 2 figs.

  10. An intercistronic region and ribosome-binding site in bacterial messenger RNA.

    PubMed Central

    Platt, T; Yanofsky, C

    1975-01-01

    A messenger RNA fragment about 220 nucleotides long has been isolated from 32-P-labeled tryptophan operon mRNA of Escherichia coli. When point mutations at the end of trpB and the beginning of trpA were introduced, the resulting nucleotide changes were found; hence the mRNA fragment must include the trpB-trpA intercistronic region. Most of the nucleotide sequences can be assigned to specific locations in the structural genes, based on the amino-acid sequences of the trpB and trpA proteins. In vitro, ribosomes bind to this piece of mRNA and protect from nuclease attack a region about 40 nucleotides long, containing a central AUG codon. The triplet codons to the 3' side of this AUG correspond to the first seven amino acids of the trpA protein; the codons to the 5' side correspond to the last six amino acids of the trpB protein. Translation of trpB is terminated by single UGA codon, which overlaps the trpA AUG initiation codon: UGAUG. Thus the untranslated "intercistronic" region consists of only two nucleotides. The RNA sequence spanning this region undoubtedly fulfills two functions, specifying ribosome recognition signals as well as encoding amino-acid sequences. Images PMID:1094468

  11. Identification of significantly mutated regions across cancer types highlights a rich landscape of functional molecular alterations

    PubMed Central

    Araya, Carlos L.; Cenik, Can; Reuter, Jason A.; Kiss, Gert; Pande, Vijay S.; Snyder, Michael P.; Greenleaf, William J.

    2015-01-01

    Cancer sequencing studies have primarily identified cancer-driver genes by the accumulation of protein-altering mutations. An improved method would be annotation-independent, sensitive to unknown distributions of functions within proteins, and inclusive of non-coding drivers. We employed density-based clustering methods in 21 tumor types to detect variably-sized significantly mutated regions (SMRs). SMRs reveal recurrent alterations across a spectrum of coding and non-coding elements, including transcription factor binding sites and untranslated regions mutated in up to ∼15% of specific tumor types. SMRs reveal spatial clustering of mutations at molecular domains and interfaces, often with associated changes in signaling. Mutation frequencies in SMRs demonstrate that distinct protein regions are differentially mutated among tumor types, as exemplified by a linker region of PIK3CA in which biophysical simulations suggest mutations affect regulatory interactions. The functional diversity of SMRs underscores both the varied mechanisms of oncogenic misregulation and the advantage of functionally-agnostic driver identification. PMID:26691984

  12. Mutational analysis of the 5' non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation.

    PubMed Central

    Parkin, N T; Cohen, E A; Darveau, A; Rosen, C; Haseltine, W; Sonenberg, N

    1988-01-01

    The first 111 nt from the 5' end of human immunodeficiency virus type 1 (HIV-1) mRNAs are shown to have a strong inhibitory effect on the translation of mRNA in in vitro translation extracts as well as in Xenopus oocytes. Mutations in the sequence of the 5' untranslated region (UTR) designed to disrupt predicted secondary structure of this region relieve the inhibition. Inhibition is restored by mutations that reconstruct the predicted secondary structure. The accessibility of the 5'-terminal cap structure was also found to be increased by some of these mutations. We conclude that secondary structure in the 5' UTR of HIV-1 mRNAs and resultant inaccessibility of the cap structure is responsible for the inhibition of translation. The implications of these findings for the understanding of the life cycle of HIV-1 are discussed. Images PMID:3181141

  13. Sequence and single-base polymorphisms of the bovine alpha-lactalbumin 5'-flanking region.

    PubMed

    Bleck, G T; Bremel, R D

    1993-04-30

    The alpha-lactalbumin (alpha LA)-encoding gene is a potential quantitative trait locus in dairy animals. In cattle, the production of alpha LA is tightly coupled to the onset of lactation and it serves as a regulatory subunit of the enzyme responsible for lactose synthesis. Lactose is the major osmole controlling water movement in the mammary gland. To better understand the control of bovine alpha LA expression, the 5'-flanking region of a Holstein alpha LA gene was cloned and sequenced. The sequenced clone contains 1952 bp of 5'-flanking region and 66-bp of the protein-coding region. Three single-bp polymorphisms were identified within this region. These polymorphisms occur at positions +15, +21 and +54 relative to the mRNA transcription start point (tsp). The +15 and +21 variations occur in the region encoding the 5'-untranslated region of the mRNA-coding sequence. The +54 polymorphism is a silent mutation in the SP-coding region of the gene. A polymerase chain reaction (PCR, Cetus)-based screening method has been employed to analyze the genotype of cattle at the +15 position. A total of 501 randomly selected cattle from seven breeds were screened for this allele. Of these animals, only the Holstein breed of cattle was found to contain the +15 variation and it occurs at a gene frequency of 32%. Sequence comparisons were conducted between the 5'-flanking regions of the bovine-milk-protein encoding genes, alpha LA, beta-casein and alpha S1-casein, which are coordinately expressed. Regions of similarity extending to 350 bp in length were observed between these sequences.

  14. 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…

  15. 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.

  16. 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.

  17. Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta- cytoplasmic actin messenger RNAs to different cytoplasmic compartments [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 2):following 1907

    PubMed Central

    1993-01-01

    We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta- actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co- hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta- galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3'UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5'UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3'UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm. PMID:8408195

  18. Host cell proteins binding to domain IV of the 5' noncoding region of poliovirus RNA.

    PubMed Central

    Blyn, L B; Chen, R; Semler, B L; Ehrenfeld, E

    1995-01-01

    Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa. PMID:7769700

  19. Allelic and haplotypic diversity of 5'promoter region of the MICA gene.

    PubMed

    Luo, Jia; Tian, Wei; Pan, FengHua; Liu, XueXiang; Li, LiXin

    2014-04-01

    In this study, the 5'promoter region of MHC class I chain-related gene A (MICA) was investigated in 104 healthy, unrelated Han individuals recruited from northern China, using PCR-sequencing method. Twelve variable sites were detected, which were in very strong linkage disequilibrium with each other. Twelve different MICA 5'promoter haplotypes were identified, among which Promoter-7 predominated (0.5529). Twenty-six extended haplotypes incorporating MICA 5'promoter and MICA exons 2-5 were observed in this population, 9 of which were in significant linkage disequilibrium (LD). Phylogenetic analysis of 5'promoter refined MICA sub-lineage structure previously constructed according to MICA coding and 3'untranslated regions. Ewens-Watterson homozygosity statistics at MICA 5'promoter region were consistent with neutral expectations. None of the five variable sites detected within the minimal promoter of MICA gene was located in the putative binding sites for transcription factor. Our study provided for the first time the sequence information about 5'promoter of MICA gene at a human population level. The data will facilitate the understanding of regulation of MICA gene expression, which represents a promising pathway for immune intervention against cancer, autoimmune disorders and infections.

  20. Modulated Binding of SATB1, a Matrix Attachment Region Protein, to the AT-Rich Sequence Flanking the Major Breakpoint Region of BCL2

    PubMed Central

    Ramakrishnan, Meera; Liu, Wen-Man; DiCroce, Patricia A.; Posner, Aleza; Zheng, Jian; Kohwi-Shigematsu, Terumi; Krontiris, Theodore G.

    2000-01-01

    The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3′ to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function. PMID:10629043

  1. Human surfactant protein-C: Genetic homogeneity and expression in RDS; comparison with other species

    SciTech Connect

    Hatzis, D.; Deiter, G.; deMello, D.E.; Floros, J. Harvard Medical School, Boston, MA Cardinal Glennon Children's Hospital, St. Louis, MO )

    1994-01-01

    Human surfactant protein C (SP-C) mRNA is detected early during fetal lung development before the differentiation of the type II cell and the need for surfactant. Later in life SP-C contributes to the surface-lowering properties of surfactant, as shown by several investigators. In this study the authors sequenced both coding and noncoding regions of 12 genomic SP-C clones from several human groups including RDS (respiratory distress syndrome), whites, and black Nigerians, and examined the expression of SP-C in tissues from RDS and from non-RDS. The data showed that all clones had identical DNA sequences, not only within coding regions, consistent with previous observations, but also within intervening, 5[prime] flanking, and 3[prime] untranslated regions. Some differences from the previously published sequence were noted. The expression of SP-C in tissues from RDS and non-RDS as determined by tissue in situ hybridization was comparable between the two groups, suggesting that altered SP-C expression, the result of pretranslational regulatory abnormalities, is an unlikely contributor to the pathogenesis of RDS. In addition the results show, using genomic blot analysis, that a remarkable conservation within coding and 5[prime] flanking but not within 3[prime] untranslated sequences exists in all mammalian species examined. These data taken together suggest that strong evolutionary pressures have been exerted on SP-C to maintain conservation, not only among humans but also among species, which may underscore important roles of SP-C in as yet unknown developmental/functional lung processes. 38 refs., 5 figs., 2 tabs.

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

    SciTech Connect

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

    1994-09-01

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

  3. 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.

  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. 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.

  6. The polycystic kidney disease 1 gene lies in a duplicated genomic region

    SciTech Connect

    Ward, C.J.; Hughes, J.; Peral, B. |

    1994-09-01

    The polycystic kidney disease 1 (PKD1) gene is situated in chromosomal band 16p13.3 and encodes a 14 kb transcript. The 5{prime} region of the PKD1 gene is located within a 40-50 kb stretch of genomic DNA which is duplicated several times in the more proximal region, 16p13.1. This proximal area gives rise to at least three transcripts designated homologous gene A (HG-A; 21 kb), HG-B (17 kb) and HG-C (8.5 kb). These three transcripts share substantial homology with each other and the PKD1 transcript. However, the 3{prime} 3.8 kb section of the PKD1 transcript is unique because it is encoded by a region of the gene that lies outside the duplicated area. The presence of the duplicate transcripts in all tissues analyzed has hampered attempts to clone and sequence the bone fide PKD1 gene. Comparison of cDNAs known to arise from the PKD1 transcript to those from the HG transcripts reveals that divergence of 2-3% has occurred between these sequences. To overcome the problem of the duplication, a large 15 kb section of genomic DNA has been sequenced together with several large HG cDNAs. Utilizing a radiation hybrid which contains only the 16p13.3 region and expresses low levels of the PKD1 transcript, we are now attempting to clone the duplicated part of the PKD1 gene by exon linking.

  7. Mapping the human growth hormone-releasing hormone receptor (GHRHR) gene to the short arm of chromosome 7(7p13-p21) near the epidermal growth factor receptor (EGFR) gene

    SciTech Connect

    Vamvakopoulos, N.C. ); Kunz, J.; Olberding, U. ); Scherer, S.W. ); Sioutopoulou, O.T. ); Schneider, V.; Durkin, A.S.; Nierman, W.C. )

    1994-03-15

    In this report, the authors have assigned the human GHRHR gene to chromosome 7p13-p21, using polymerase chain reaction (PCR) amplification of DNA from well-defined human-rodent somatic cell hybrids. The GHRHR gene was assigned to human chromosome 7 by discordancy analysis (data not shown) of PCR amplification products from NIGMS mapping panel Nos. 1 and 2 DNA templates. The PCR primers (p[sub f], 5[prime]-GCTGCCTCATCACGCCACTGGAGTCCAC-3[prime]; and P[sub r], 5[prime]-CAGGTTTATTGGCTCCTCTGAGCCTTGG-3[prime]) amplified a 276-bp-long fragment from the 3[prime] untranslated region of the human GHRHR gene. Subsequently, they determined the location of the GHRHR gene within human chromosome 7 by PCR amplification of genomic DNA template from somatic cell hybrids that contain deletions of this chromosome. Amplification of the 276-bp DNA fragment was seen only in the cell lines that contained an intact chromosome 7 short arm. The lack of amplification using genomic DNA from 0044 Rag 1-15 and It A9 2-21-14 maps this gene to 7p13-p21. Additionally, the appropriate amplified product was observed from the human chromosome 4 containing NIGMS panel 2 cell line GM10115. This line was reported to have retained a small region of human chromosome 7 containing the epidermal growth factor receptor (EGFR) gene that is mapped to 7p12-p13. The authors conclude that the human GHRHR gene maps to the small arm of chromosome 7 within 7p13-p21 and close to the EGFR gene. This assignment is consistent with the syntenic relationship between mouse chromosome 6 and human chromosome 7 in this region.

  8. Ephemeral regions versus pseudo ephemeral regions

    NASA Technical Reports Server (NTRS)

    Martin, S. F.; Livi, S. H. B.; Wang, J.; Shi, Z.

    1985-01-01

    New studies of the quiet Sun reveal that ephemeral active regions constitute minority rather than a majority of all the short lived, small scale bipolar features on the Sun. In contrast to the recognized patterns of growth and decay of ephemeral regions, various examples of the creation of other temporary bipoles nicknamed pseudo ephemeral regions are illustrated. The pseudo ephemeral regions are the consequence of combinations of small scale dynamic processes of the quiet Sun including: (1) fragmentation of network magnetic fields, (2) the separation of opposite polarity halves of ephemeral regions as they grow and evolve, and (3) the coalescence of weak network or intranetwork magnetic fields. New observations offer the possibility of resolving the discrepancies that have arisen in the association of ephemeral regions with X-ray bright points. Many X-ray bright points may be related to those pseudo ephemeral regions which have begun to exhibit magnetic flux loss.

  9. The human myelin oligodendrocyte glycoprotein (MOG) gene: Complete nucleotide sequence and structural characterization

    SciTech Connect

    Paule Roth, M.; Malfroy, L.; Offer, C.; Sevin, J.; Enault, G.; Borot, N.; Pontarotti, P.; Coppin, H.

    1995-07-20

    Human myelin oligodendrocyte glycoprotein (MOG), a myelin component of the central nervous system, is a candidate target antigen for autoimmune-mediated demyelination. We have isolated and sequenced part of a cosmid clone that contains the entire human MOG gene. The primary nuclear transcript, extending from the putative start of transcription to the site of poly(A) addition, is 15,561 nucleotides in length. The human MOG gene contains 8 exons, separated by 7 introns; canonical intron/exon boundary sites are observed at each junction. The introns vary in size from 242 to 6484 bp and contain numerous repetitive DNA elements, including 14 Alu sequences within 3 introns. Another Alu element is located in the 3{prime}-untranslated region of the gene. Alu sequences were classified with respect to subfamily assignment. Seven hundred sixty-three nucleotides 5{prime} of the transcription start and 1214 nucleotides 3{prime} of the poly(A) addition sites were also sequenced. The 5{prime}-flanking region revealed the presence of several consensus sequences that could be relevant in the transcription of the MOG gene, in particular binding sites in common with other myelin gene promoters. Two polymorphic intragenic dinucleotide (CA){sub n} and tetranucleotide (TAAA){sub n} repeats were identified and may provide genetic marker tools for association and linkage studies. 50 refs., 3 figs., 3 tabs.

  10. 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

  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. An unusually long non-coding region in rat lens alpha-crystallin messenger RNA.

    PubMed Central

    Moormann, R J; van der Velden, H M; Dodemont, H J; Andreoli, P M; Bloemendal, H; Schoenmakers, J G

    1981-01-01

    Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs. Images PMID:6171772

  13. 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. PMID:17691096

  14. 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...

  15. 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.

  16. Expression of adenovirus-2 early region 4: assignment of the early region 4 polypeptides to their respective mRNAs, using in vitro translation.

    PubMed Central

    Tigges, M A; Raskas, H J

    1982-01-01

    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

  17. 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…

  18. An Origin of DNA Replication in the Promoter Region of the Human Fragile X Mental Retardation (FMR1) Gene▿ †

    PubMed Central

    Gray, Steven J.; Gerhardt, Jeannine; Doerfler, Walter; Small, Lawrence E.; Fanning, Ellen

    2007-01-01

    Fragile X syndrome, the most common form of inherited mental retardation in males, arises when the normally stable 5 to 50 CGG repeats in the 5′ untranslated region of the fragile X mental retardation protein 1 (FMR1) gene expand to over 200, leading to DNA methylation and silencing of the FMR1 promoter. Although the events that trigger local CGG expansion remain unknown, the stability of trinucleotide repeat tracts is affected by their position relative to an origin of DNA replication in model systems. Origins of DNA replication in the FMR1 locus have not yet been described. Here, we report an origin of replication adjacent to the FMR1 promoter and CGG repeats that was identified by scanning a 35-kb region. Prereplication proteins Orc3p and Mcm4p bind to chromatin in the FMR1 initiation region in vivo. The position of the FMR1 origin relative to the CGG repeats is consistent with a role in repeat maintenance. The FMR1 origin is active in transformed cell lines, fibroblasts from healthy individuals, fibroblasts from patients with fragile X syndrome, and fetal cells as early as 8 weeks old. The potential role of the FMR1 origin in CGG tract instability is discussed. PMID:17101793

  19. 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.

  20. 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.

  1. 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.

  2. Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-{alpha}-treated A549 cells

    SciTech Connect

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou; Huang, Xin; Wang, Tao; Huang, Xiao; Li, Han; Zhang, Luyong

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Triptolide inhibited COX-2 expression and the half-life of COX-2 mRNA is decreased. Black-Right-Pointing-Pointer The HuR protein shuttling from nucleus to cytoplasm is inhibited by triptolide. Black-Right-Pointing-Pointer Triptolide inhibited 3 Prime -UTR fluorescence reporter gene activity. Black-Right-Pointing-Pointer COX-2 mRNA binding to HuR is decreased by triptolide in pull-down experiments. -- Abstract: Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-{alpha}-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3 Prime -untranslated region (3 Prime -UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

  3. MiR-214 inhibits cell growth in hepatocellular carcinoma through suppression of {beta}-catenin

    SciTech Connect

    Wang, Xiaojun; Chen, Ji; Li, Feng; Lin, Yanting; Zhang, Xiaoping; Lv, Zhongwei; Jiang, Jiaji

    2012-11-30

    Highlights: Black-Right-Pointing-Pointer miR-214 is frequently downregulated in human HCC cell lines and tissues. Black-Right-Pointing-Pointer miR-214 overexpression inhibits HCC cell growth in vitro and in vivo. Black-Right-Pointing-Pointer miR-214 directly targets {beta}-catenin 3 Prime -UTR in HCC cells. Black-Right-Pointing-Pointer miR-214 regulates {beta}-catenin downstream signaling molecules. -- Abstract: Mounting evidence has shown that microRNAs (miRNAs) are implicated in carcinogenesis and can function as oncogenes or tumor suppressor genes in human cancers. Recent profile studies of miRNA expression have documented a deregulation of miRNA (miR-214) in hepatocellular carcinoma (HCC). However, its potential functions and underlying mechanisms in hepatocarcinogenesis remain largely unknown. Here, we confirmed that miR-214 is significantly downregulated in HCC cells and specimens. Ectopic overexpression of miR-214 inhibited proliferation of HCC cells in vitro and tumorigenicity in vivo. Further studies revealed that miR-214 could directly target the 3 Prime -untranslated region (3 Prime -UTR) of {beta}-catenin mRNA and suppress its protein expression. Similar to the restoring miR-214 expression, {beta}-catenin downregulation inhibited cell growth, whereas restoring the {beta}-catenin expression abolished the function of miR-214. Moreover, miR-214-mediated reduction of {beta}-catenin resulted in suppression of several downstream genes including c-Myc, cyclinD1, TCF-1, and LEF-1. These findings indicate that miR-214 serves as tumor suppressor and plays substantial roles in inhibiting the tumorigenesis of HCC through suppression of {beta}-catenin. Given these, miR-214 may serve as a useful prognostic or therapeutic target for treatment of HCC.

  4. Utah: Salt Lake Region

    Atmospheric Science Data Center

    2014-05-15

    article title:  Winter and Summer Views of the Salt Lake Region     View Larger Image Magnificent views of the region surrounding Salt Lake City, Utah are captured in these winter and summer images from the ...

  5. [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.

  6. 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 their…

  7. Regional flood frequency analysis

    SciTech Connect

    Singh, V.P.

    1987-01-01

    This book, the fourth of a four volume set, contains five sections encompassing major aspects of regional flood frequency analysis. Each section starts usually with an invited state-of-the-art paper followed by contributed papers. The first section provides an assessment of regional flood frequency analysis. Methods for performing regional frequency analysis for ungaged watersheds are presented in Section 2. More discussion on regional frequency analysis is provided in Section 3. Selection and comparison of regional frequency methods are dealt with in Section 4; these are of great interest to the user. Increasing attention is being focused these days on paleohydrologic flood analysis. This topic is covered in Section 5.

  8. Reversed-polarity regions

    NASA Technical Reports Server (NTRS)

    Tang, F.

    1982-01-01

    It is found by a statistical study of 58 reversed-polarity regions (RPRs) covering the 11-year period 1969-1979 that RPRs (1) have a lifespan comparable to normal active regions, (2) do not show a tendency to rotate toward a more normal alignment, and (3) have stable configurations that do not suggest stress due to their anomalous magnetic alignment. As in normal regions, RPR magnetic complexity is found to be the primary factor in flare productivity. Weak-field RPRs produce no flares, and regions with complex spots produce more flares than regions with non-complex spots by a factor of five. The main difference between RPRs and normal regions lies in complex spot frequency, with less that 17% of normal active regions having such spots and fewer than 1.8% having long-lived complex ones, while 41% of RPRs have complex spots and 24% have long-lived complex spots.

  9. The crisis of regionalization.

    PubMed

    Marchildon, Gregory P

    2015-11-01

    Currently in Canada, there is no consensus concerning the efficacy of regionalization, a reversal of the strong commitment in favour only a decade earlier. Instead, provincial governments are either dismantling regional health authorities in favour of highly centralized structures under the control of ministries of health or actively considering more centralized approaches. There is a general feeling among political leaders that regionalization has failed to achieve its original objectives. However, by not including physicians and primary care within regionalized governance, provincial governments have never given regionalization a real chance. Moreover, given the fact that the status quo prior to regionalization was far from an ideal state and would be almost impossible to return to in any event, some provincial governments should consider implementing a more full-blooded version of regionalization before abandoning the approach.

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

    SciTech Connect

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

    1994-11-15

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

  11. Isolation and characterization of two overlapping cosmid clones from the 4q35 region, near the facioscapulohumeral muscular dystrophy locus

    SciTech Connect

    Deidda, G.; Grisanti, P.; Vigneti, E.

    1994-09-01

    The gene for facioscapulohumeral muscular dystrophy (FSHD) has been localized by linkage analysis to the 4q35 region. The most telomeric p13E-11 prove has been shown to detect 4q35 DNA rearrangements in both sporadic and familial cases of the disease. With the aim of constructing a detailed physical map of the 4q35 region and searching for the mutant gene, we used p13E-11 probe to isolate cosmid clones from a human genomic library in a pCos-EMBL 2 vector. Two positive clones were isolated, clones 3 and 5, which partially overlap and carry human genomic inserts of 42 and 45 kb, respectively. The cosmids share a common region containing the p13E-11 region and a stretch of KpnI units consisting of 3.2 kb tandemly repeated sequences (about 10). The restriction maps were constructed using the following enzymes: Bam HI, BgIII, Eco RI, EcoRV, KpnI and Sfi I. Clone 3 extends 4 kb upstream of C5 and stops within the Kpn repeats. Clone 5 extends 4 kb downstream from the Kpn repeats and it presents an additional EcoRI site. Clone 5 contains a stretch of Kpn sequences of nearly 32 kb, corresponding to 10 Kpn repeats; clone 3 contains a stretch of 29 kb corresponding to 9 Kpn repeats, as determined by PFGE analysis of partial digestion of the clones. Clone 5 seems to contain the entire Eco RI region prone to rearrangements in FSHD patients. From clone 5 several subclones were obtained, from the Kpn region and from the region spanning from the last Kpn repeat to the cloning site. No single copy sequences were detected. Subclones from the 3{prime} end region contain beta-satellite or Sau3A-like sequences. In situ hybridization with the whole C5 cosmid shows hybridization signals at the tip of chromosome 4 (4q35) and chromosome 10 (10q26), in the pericentromeric region of chromosome 1 (1q12) and in the p12 region of the acrocentric chromosomes (chr. 21, 22, 13, 14, 15).

  12. Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

    PubMed

    Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

    1989-03-01

    The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted

  13. Amplification of GB virus-C/hepatitis G virus RNA with primers from different regions of the viral genome.

    PubMed

    Kao, J H; Chen, P J; Chen, W; Hsiang, S C; Lai, M Y; Chen, D S

    1997-04-01

    GB virus-C/hepatitis G virus (GBV-C/HGV) is a newly identified RNA virus. The aim of the study was to compare three primer pairs from the 5' untranslated region (5'UTR), envelope region 2 (E 2) and nonstructural region 3 (NS 3) of GBV-C/HGV genome for their ability to detect GBV-C/HGV RNA by polymerase chain reaction (PCR) assays. By using PCR with primers from different regions of the viral genome, serum GBV-C/HGV RNA was assayed in 200 at-risk individuals. The sensitivity of this assay was assessed by a titration experiment, and nucleotide sequences of the amplified products were determined directly. Of 200 serum samples, 43 (21.5%) were positive for GBV-C/HGV RNA with at least one of the primer pairs. The positive rates by 5'UTR, NS 3, and E 2 primers were 100%, 98%, and 84%, respectively, and the sensitivity of PCR assays using 5'UTR primers was 10 to 100 times more likely to detect GBV-C/HGV RNA than that of NS 3 and E 2 primers. The average homology of amplified targets to the prototype HGV genome was 89%, 80%, and 85% and the similarity between each amplified target was up to 100%, 90%, and 92% in the 5'UTR, E 2, and NS 3 regions, respectively. Therefore, the 5'UTR of GBV-C/HGV genome is highly conserved and primers deduced from this region can provideva sensitive and specific PCR assay for GBV-C/HGV RNA.

  14. Characterization of the Promoter Regions of Two Sheep Keratin-Associated Protein Genes for Hair Cortex-Specific Expression.

    PubMed

    Zhao, Zhichao; Liu, Guangbin; Li, Xinyun; Huang, Ji; Xiao, Yujing; Du, Xiaoyong; Yu, Mei

    2016-01-01

    The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3-3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5' flanking/5' untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5' upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays. PMID:27100288

  15. Characterization of the Promoter Regions of Two Sheep Keratin-Associated Protein Genes for Hair Cortex-Specific Expression

    PubMed Central

    Zhao, Zhichao; Liu, Guangbin; Li, Xinyun; Huang, Ji; Xiao, Yujing; Du, Xiaoyong; Yu, Mei

    2016-01-01

    The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3–3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5′ flanking/5′ untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5′ upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays. PMID:27100288

  16. Analysis of Polymorphisms and Haplotype Structure of the Human Thymidylate Synthase Genetic Region: A Tool for Pharmacogenetic Studies

    PubMed Central

    Ghosh, Soma; Hossain, M. Zulfiquer; Borges, Michael; Goggins, Michael G.; Ingersoll, Roxann G.; Eshleman, James R.; Klein, Alison P.; Kern, Scott E.

    2012-01-01

    5-fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5′-untranslated region (5′-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing. PMID:22496803

  17. Characterization of the Promoter Regions of Two Sheep Keratin-Associated Protein Genes for Hair Cortex-Specific Expression.

    PubMed

    Zhao, Zhichao; Liu, Guangbin; Li, Xinyun; Huang, Ji; Xiao, Yujing; Du, Xiaoyong; Yu, Mei

    2016-01-01

    The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3-3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5' flanking/5' untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5' upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays.

  18. Molecular cloning of rhamnose-binding lectin gene and its promoter region from snakehead Channa argus.

    PubMed

    Jia, W Z; Shang, N; Guo, Q L

    2010-09-01

    Lectins are sugar-binding proteins that mediate pathogen recognition and cell-cell interactions. A rhamnose-binding lectin (RBL) gene and its promoter region have been cloned and characterized from snakehead Channa argus. From the transcription initiation site, snakehead rhamnose-binding lectin (SHL) gene extends 2,382 bp to the end of the 3' untranslated region (UTR), and contains nine exons and eight introns. The open reading frame (ORF) of the SHL transcript has 675 bp which encodes 224 amino acids. The molecular structure of SHL is composed of two tandem repeat carbohydrate recognition domains (CRD) with 35% internal identity. Analysis of the gene organization of SHL indicates that the ancestral gene of RBL may diverge and evolve by exon shuffling and gene duplication, producing new forms to play their own roles in various organisms. The characteristics of SHL gene 5' flanking region are the presence of consensus nuclear factor of interleukin 6 (NF-IL6) and IFN-gamma activation (GAS) sites. The results provide indirect evidence that up-regulation of SHL expression may be induced in response to inflammatory stimuli, such as lipopolysaccharide (LPS), interleukin 6 (IL-6), and interferon gamma (IFN-gamma). The transcript of SHL mRNA was expressed in the head kidney, posterior kidney, spleen, liver, intestine, heart, muscle, and ovary. No tissue-specific expressive pattern is different from reported STLs, WCLs, and PFLs, suggesting that different types of RBLs exist in species-specific fish that have evolved and adapted to their surroundings.

  19. Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

    NASA Technical Reports Server (NTRS)

    Shibata, K.; Abe, S.; Davies, E.

    2001-01-01

    Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

  20. Deletions of a differentially methylated CpG island at SNRPN define a putative imprinting control region

    SciTech Connect

    Sutcliffe, J.S.,; Nakao, M.; Beaudet, A.L.

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies, respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy, or other mutations. Four transcripts designated PAR-5, PAR-7, PAR-1 and PAR-4 were isolated and localized to a region within 300 kb telomeric to the gene encoding small nuclear ribonucleoprotein-associated polypeptide N (SNRPN). Analysis of the transcripts in cultured fibroblasts and lymphoblasts from deletion patients demonstrated that SNRPN, PAR-5 and PAR-1 are expressed exclusively from the paternal chromosome, defining an imprinted domain that spans at least 200 kb. All three imprinted transcripts were absent in cells from three PWS patients (one pair of sibs and one sporadic case) with small deletions that involve a differentially methylated CpG island containing a previously undescribed 5{prime} untranslated exon ({alpha}) of SNRPN. Methylation of the CpG island is specific for the maternal chromosome consistent with paternal expression of the imprinted domain. One deletion, which is benign when maternally transmitted, extends upstream <30 kb from the CpG island, and is associated with altered methylation centromeric to SNRPN, and loss of transcription telomeric to SNRPN, implying the presence of an imprinting control region around the CpG island containing exon {alpha}.

  1. Ground- and excited-state tautomerism in 2-(3{prime}-hydroxy-2{prime}-pyridyl)benzimidazole

    SciTech Connect

    Prieto, F.R.; Rodriguez, M.C.R.; Gonzalez, M.M.; Fernandez, M.A.R.

    1994-09-01

    Ground-state HPyBI is determined to have keto-enol equilibrium in water, and the enol form predominates in nonaqueous solutions. The keto form is the only excited form in all the solvents considered. Ultrafast intramolecular proton transfer creates the enol form from the keto form. 47 refs., 6 figs., 3 tabs.

  2. Conformational analysis of four spiro6cyclohexane-1,3prime;-indolin9-2prime;-one derivatives

    NASA Astrophysics Data System (ADS)

    Halász, Judit; Podányi, Benjamin; Sánta-Csutor, Andrea; Böcskei, Zsolt; Simon, Kálmán; Hanusz, Miklós; Hermecz, István

    2003-06-01

    SR 121463 is a potent and selective, orally active vasopressin V 2 receptor antagonist. During the synthesis of SR 121463, the formation of the stereochemistry of the cyclohexyl moiety is one of the most important steps. Conformational analysis (via NMR studies and, for cis- 3, also via X-ray structure determination) of the isomers obtained in this step is reported.

  3. REGIONAL EMAP PROPOSALS

    EPA Science Inventory

    The US EPA's Environmental Assessment and Monitoring Program (EMAP) annually funds regional EMAP (REMAP) projects through each of the regions to support the improvement of monitoring activities by the states. The last call for proposals emphasized the need to support biological m...

  4. New Lessons in Regionalism.

    ERIC Educational Resources Information Center

    Peirce, Neal R.

    1998-01-01

    From the hierarchical, governmental, industrial, military model of the 20th century, society is moving rapidly toward a globalized, interactive market. This new order is tailor-made for regional development but also challenges regions to think and act strategically in a world of weakened central governments and growing international currency…

  5. Ad Hoc Rural Regionalism

    ERIC Educational Resources Information Center

    Hamin, Elisabeth M.; Marcucci, Daniel J.

    2008-01-01

    A new regionalism has been much documented and researched for metropolitan areas; this article documents that there is a new rural regionalism as well. In the United States, these groups appear most likely to emerge in areas that are challenged by outcomes characterizing globalization's effects on the rural condition: namely, exurban or…

  6. Regional anesthesia for laparoscopy.

    PubMed

    Collins, L M; Vaghadia, H

    2001-03-01

    A variety of laparoscopic procedures can be performed on patients under regional anesthesia. Diagnostic laparoscopy in elective and emergency patients, pain mapping, laparoscopy for infertility, and tubal sterilization are some examples. The key benefits of regional anesthesia include less emesis, less postoperative pain, shorter postoperative stay, improved patient satisfaction, and overall safety. Regional techniques, such as rectus sheath blocks, inguinal blocks, and caudal blocks, are useful adjuncts to general anesthesia and facilitate postoperative analgesia. Other techniques, such as spinal and epidural anesthesia, and combination of the two, are suitable as a sole anesthetic technique for laparoscopy. The physiologic changes during laparoscopy in the awake patient appear to be tolerated well under regional anesthesia. It is reasonable to assume that with advances in instrumentation and surgical techniques, the role of laparoscopy will increase in the future. The benefits conferred by regional anesthesia make it an attractive option to general anesthesia for many patients and procedures. Successful implementation of regional anesthesia is an important determinant of how anesthesiologists, surgeons, and surgical facilities cope with new challenges. In the future, it could be possible to provide "walk-in/walk-out" regional anesthesia with a real possibility of fast tracking patients through the recovery process after ambulatory surgery. For maximal patient safety, however, facilities offering regional anesthesia must have appropriately trained anesthesia personnel and the equipment necessary for monitoring and providing full resuscitation in the event of complications or a need to convert to general anesthesia. PMID:11244919

  7. South Persian Gulf Region

    NASA Technical Reports Server (NTRS)

    1981-01-01

    This oblique view of the south Persian Gulf region (26.0N, 54.0E) was taken over Iran looking west across the south Persian Gulf into the Trucial Coast of the United Arab Emirates and the prominent Qatar peninsula. Rich in petroleum resources, this region supplies much of the world's oil needs from its many ports and off shore loading facilities.

  8. Reversed-polarity regions

    NASA Technical Reports Server (NTRS)

    Tang, F.

    1980-01-01

    The 58 RPRS studied have a lifespan comparable to normal active regions and have no tendency to rotate toward a more normal alignment. They seem to have stable configurations with no apparent evidence suggesting stress due to their anomalous magnetic alignment. Magnetic complexity in RPRs is the key to flare productivity just as it is in normal regions - weak field RPRs produced no flares and regions with complex spots produced more flares than regions with noncomplex spots by a factor of 5. The RPRs however, differ from normal regions in the frequency of having complex spots, particularly the long lived complex spots, in them. Less than 17 percent of normal ARs have complex spots; less than 1.8 percent have long lived complex spots. In contrast, 41 percent of RPRs have complex spots and 24 percent have long lived complex spots.

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

    SciTech Connect

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

    1988-06-01

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

  10. Modeling the transition region

    NASA Technical Reports Server (NTRS)

    Singer, Bart A.

    1993-01-01

    The current status of transition-region models is reviewed in this report. To understand modeling problems, various flow features that influence the transition process are discussed first. Then an overview of the different approaches to transition-region modeling is given. This is followed by a detailed discussion of turbulence models and the specific modifications that are needed to predict flows undergoing laminar-turbulent transition. Methods for determining the usefulness of the models are presented, and an outlook for the future of transition-region modeling is suggested.

  11. Regional ocean data assimilation.

    PubMed

    Edwards, Christopher A; Moore, Andrew M; Hoteit, Ibrahim; Cornuelle, Bruce D

    2015-01-01

    This article reviews the past 15 years of developments in regional ocean data assimilation. A variety of scientific, management, and safety-related objectives motivate marine scientists to characterize many ocean environments, including coastal regions. As in weather prediction, the accurate representation of physical, chemical, and/or biological properties in the ocean is challenging. Models and observations alone provide imperfect representations of the ocean state, but together they can offer improved estimates. Variational and sequential methods are among the most widely used in regional ocean systems, and there have been exciting recent advances in ensemble and four-dimensional variational approaches. These techniques are increasingly being tested and adapted for biogeochemical applications.

  12. Identification of a cis-acting replication element within the poliovirus coding region.

    PubMed

    Goodfellow, I; Chaudhry, Y; Richardson, A; Meredith, J; Almond, J W; Barclay, W; Evans, D J

    2000-05-01

    The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

  13. Complex Regional Pain Syndrome

    MedlinePlus

    Complex regional pain syndrome (CRPS) is a chronic pain condition. It causes intense pain, usually in the arms, hands, legs, or feet. ... in skin temperature, color, or texture Intense burning pain Extreme skin sensitivity Swelling and stiffness in affected ...

  14. Mercury's South Polar Region

    NASA Video Gallery

    This animation shows 89 wide-angle camera (WAC) images of Mercury’s south polar region acquired by the Mercury Dual Imaging System (MDIS) over one complete Mercury solar day (176 Earth days). Thi...

  15. Regional Instrumentation Centers.

    ERIC Educational Resources Information Center

    Cromie, William J.

    1980-01-01

    Focuses on the activities of regional instrumentation centers that utilize the state-of-the-art instruments and methodology in basic scientific research. The emphasis is on the centers involved in mass spectroscopy, magnetic resonance spectroscopy, lasers, and accelerators. (SA)

  16. On regional geomagnetic charts

    USGS Publications Warehouse

    Alldredge, L.R.

    1987-01-01

    When regional geomagnetic charts for areas roughly the size of the US were compiled by hand, some large local anomalies were displayed in the isomagnetic lines. Since the late 1960s, when the compilation of charts using computers and mathematical models was started, most of the details available in the hand drawn regional charts have been lost. One exception to this is the Canadian magnetic declination chart for 1980. This chart was constructed using a 180 degrees spherical harmonic model. -from Author

  17. Physical mapping of chromosome 17p13.3 in the region of a putative tumor suppressor gene important in medulloblastoma

    SciTech Connect

    McDonald, J.D.; Daneshvar, L.; Willert, J.R.

    1994-09-01

    Deletion mapping of a medulloblastoma tumor panel revealed loss of distal chromosome 17p13.3 sequences in tumors from 14 of 32 patients (44%). Of the 14 tumors showing loss of heterozygosity by restriction fragment length polymorphism analysis, 14 of 14 (100%) displayed loss of the telomeric marker p144-D6 (D17S34), while a probe for the ABR gene on 17p13.3 was lost in 7 of 8 (88%) informative cases. Using pulsed-field gel electrophoresis, we localized the polymorphic marker (VNTR-A) of the ABR gene locus to within 220 kb of the p144-D6 locus. A cosmid contig constructed in this region was used to demonstrate by fluorescence in situ hybridization that the ABR gene is oriented transcriptionally 5{prime} to 3{prime} toward the telomere. This report provides new physical mapping data for the ABR gene, which has not been previously shown to be deleted in medulloblastoma. These results provide further evidence for the existence of a second tumor suppressor gene distinct from p53 on distal chromosome 17p. 12 refs., 3 figs.

  18. Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion

    PubMed Central

    Ramsey, Meghan E.; Bender, Tobias; Klimowicz, Amy K.; Hackett, Kathleen T.; Yamamoto, Ami; Jolicoeur, Adrienne; Callaghan, Melanie M.; Wassarman, Karen M.; van der Does, Chris; Dillard, Joseph P.

    2015-01-01

    Summary Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5’ untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem-loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational, and post-translational mechanisms controlling T4SS gene expression and DNA secretion. PMID:26076069

  19. Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

    PubMed

    Ramsey, Meghan E; Bender, Tobias; Klimowicz, Amy K; Hackett, Kathleen T; Yamamoto, Ami; Jolicoeur, Adrienne; Callaghan, Melanie M; Wassarman, Karen M; van der Does, Chris; Dillard, Joseph P

    2015-09-01

    Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.

  20. Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

    PubMed

    Ramsey, Meghan E; Bender, Tobias; Klimowicz, Amy K; Hackett, Kathleen T; Yamamoto, Ami; Jolicoeur, Adrienne; Callaghan, Melanie M; Wassarman, Karen M; van der Does, Chris; Dillard, Joseph P

    2015-09-01

    Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion. PMID:26076069

  1. In situ optical sequencing and structure analysis of a trinucleotide repeat genome region by localization microscopy after specific COMBO-FISH nano-probing

    NASA Astrophysics Data System (ADS)

    Stuhlmüller, M.; Schwarz-Finsterle, J.; Fey, E.; Lux, J.; Bach, M.; Cremer, C.; Hinderhofer, K.; Hausmann, M.; Hildenbrand, G.

    2015-10-01

    Trinucleotide repeat expansions (like (CGG)n) of chromatin in the genome of cell nuclei can cause neurological disorders such as for example the Fragile-X syndrome. Until now the mechanisms are not clearly understood as to how these expansions develop during cell proliferation. Therefore in situ investigations of chromatin structures on the nanoscale are required to better understand supra-molecular mechanisms on the single cell level. By super-resolution localization microscopy (Spectral Position Determination Microscopy; SPDM) in combination with nano-probing using COMBO-FISH (COMBinatorial Oligonucleotide FISH), novel insights into the nano-architecture of the genome will become possible. The native spatial structure of trinucleotide repeat expansion genome regions was analysed and optical sequencing of repetitive units was performed within 3D-conserved nuclei using SPDM after COMBO-FISH. We analysed a (CGG)n-expansion region inside the 5' untranslated region of the FMR1 gene. The number of CGG repeats for a full mutation causing the Fragile-X syndrome was found and also verified by Southern blot. The FMR1 promotor region was similarly condensed like a centromeric region whereas the arrangement of the probes labelling the expansion region seemed to indicate a loop-like nano-structure. These results for the first time demonstrate that in situ chromatin structure measurements on the nanoscale are feasible. Due to further methodological progress it will become possible to estimate the state of trinucleotide repeat mutations in detail and to determine the associated chromatin strand structural changes on the single cell level. In general, the application of the described approach to any genome region will lead to new insights into genome nano-architecture and open new avenues for understanding mechanisms and their relevance in the development of heredity diseases.

  2. Northeast Regional Biomass Program

    SciTech Connect

    Lusk, P.D.

    1992-12-01

    The Northeast Regional Biomass Program has been in operation for a period of nine years. During this time, state managed programs and technical programs have been conducted covering a wide range of activities primarily aim at the use and applications of wood as a fuel. These activities include: assessments of available biomass resources; surveys to determine what industries, businesses, institutions, and utility companies use wood and wood waste for fuel; and workshops, seminars, and demonstrations to provide technical assistance. In the Northeast, an estimated 6.2 million tons of wood are used in the commercial and industrial sector, where 12.5 million cords are used for residential heating annually. Of this useage, 1504.7 mw of power has been generated from biomass. The use of wood energy products has had substantial employment and income benefits in the region. Although wood and woodwaste have received primary emphasis in the regional program, the use of municipal solid waste has received increased emphasis as an energy source. The energy contribution of biomass will increase as potentia users become more familiar with existing feedstocks, technologies, and applications. The Northeast Regional Biomass Program is designed to support region-specific to overcome near-term barriers to biomass energy use.

  3. LAMPF transition region

    SciTech Connect

    Sander, O.R.

    1982-06-01

    After describing the transition region between the LAMPF drift-tube linac and side-coupled linac, we discuss the function of the region, its present shortcomings, and the need for a redesign. Then we present the new design, its advantages, and its expected performance. Included are detailed results of beam-dynamics studies giving the ranges of input- and output-beam shapes that can be successfully matched in the new transition region. To improve the present operation of the two linacs, we suggest small changes that will allow us to easily match the beam between the two linacs. Finally we describe the methods used in our beam-dynamic studies so that effects of future improvements to the new design can be examined.

  4. Turbulence in HII regions

    NASA Astrophysics Data System (ADS)

    O'dell, C. R.

    1986-10-01

    It has been known for many decades that the Reynolds number in HII regions must be very high and that the corresponding fine scale flow must be turbulent. Even though the theoretical relation between turbulent element separation and random velocity was derived by Kolmogoroff over forty years ago, there have been only a few attempts to test this theory and its corresponding assumptions. An attempt by Munch for M42 with marginal velocity resolution lead to ambiguous results, although more recent studies by Jean Rene Roy and his colleagues have been more credible. The internal velocities of a number of HII regions were systematically studied and the theory was tested with considerable certainty. The results should be important for the determination of the energy balance of HII regions and the relation of small scale motion to the process of star formation.

  5. Northeast Regional Biomass Program

    SciTech Connect

    O'Connell, R.A.

    1991-11-01

    The management structure and program objectives for the Northeast Regional Biomass Program (NRBP) remain unchanged from previous years. Additional funding was provided by the Bonneville Power Administration Regional Biomass Program to continue the publication of articles in the Biologue. The Western Area Power Administration and the Council of Great Lakes Governors funded the project Characterization of Emissions from Burning Woodwaste''. A grant for the ninth year was received from DOE. The Northeast Regional Biomass Steering Committee selected the following four projects for funding for the next fiscal year. (1) Wood Waste Utilization Conference, (2) Performance Evaluation of Wood Systems in Commercial Facilities, (3) Wood Energy Market Utilization Training, (4) Update of the Facility Directory.

  6. NV PFA Regional Data

    SciTech Connect

    James Faulds

    2015-10-28

    This project focused on defining geothermal play fairways and development of a detailed geothermal potential map of a large transect across the Great Basin region (96,000 km2), with the primary objective of facilitating discovery of commercial-grade, blind geothermal fields (i.e. systems with no surface hot springs or fumaroles) and thereby accelerating geothermal development in this promising region. Data included in this submission consists of: structural settings (target areas, recency of faulting, slip and dilation potential, slip rates, quality), regional-scale strain rates, earthquake density and magnitude, gravity data, temperature at 3 km depth, permeability models, favorability models, degree of exploration and exploration opportunities, data from springs and wells, transmission lines and wilderness areas, and published maps and theses for the Nevada Play Fairway area.

  7. MicroRNA-101 mediates the suppressive effect of laminar shear stress on mTOR expression in vascular endothelial cells

    SciTech Connect

    Chen, Kui; Fan, Wendong; Wang, Xing; Ke, Xiao; Wu, Guifu; Hu, Chengheng

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Laminar shear stress upregulates miR-101 expression in vascular endothelial cells. Black-Right-Pointing-Pointer miR-101 represses mTOR expression through a specific 3 Prime UTR binding site. Black-Right-Pointing-Pointer Overexpression of miR-101 inhibits G1/S transition and endothelial cell proliferation. Black-Right-Pointing-Pointer Blockade of miR-101 attenuates the suppressive effect of laminar flow on mTOR expression. -- Abstract: Shear stress associated with blood flow plays an important role in regulating gene expression and cell function in endothelial cells (ECs). MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that negatively regulate the expression of target genes by binding to the mRNA 3 Prime -untranslated region (3 Prime UTR) at the posttranscriptional level involved in diverse cellular processes. This study demonstrates that microRNA-101 in response to laminar shear stress (LSS) is involved in the flow regulation of gene expression in ECs. qRT-PCR analysis showed that miR-101 expression was significantly upregulated in human umbilical vein endothelial cells (HUVECs) exposed to 12 dyn/cm{sup 2} laminar shear stress for 12 h. We found that transfection of miR-101 significantly decreased the luciferase activity of plasmid reporter containing the 3 Prime UTR of mammalian target of rapamycin (mTOR) gene. Western analysis revealed that the protein level of mTOR was significantly reduced in ECs transfected with miR-101. Furthermore, miR-101 overexpression induced cell cycle arrest at the G1/S transition and suppressed endothelial cell proliferation. Finally, transfection of miR-101 inhibitors attenuated the suppressive effects of LSS on mTOR expression, which identified the efficacy of loss-of-function of miR-101 in laminar flow-treated ECs. In conclusion, we have demonstrated that upregulation of miR-101 in response to LSS contributes to the suppressive effects of LSS on mTOR expression and EC

  8. ORIC central region calculations

    SciTech Connect

    Bailey, J.D.; Dowling, D.T.; Lane, S.N.; Mosko, S.W.; Olsen, D.K.; Tatum, B.A.

    1995-12-31

    The central region for the K = 100 Oak Ridge Isochronous Cyclotron, ORIC, will be modified to provide better orbit centering, focusing of orbits in the axial direction, and phase selection, in order to improve extraction efficiency, and reduce radioactive activation of cyclotron components. The central region is specifically designed for the acceleration of intense light ion beams such as 60 MeV protons and 15--100 MeV alphas. These beams will be used in the production of radioactive atoms in the Radioactive Ion Beam Project at Oak Ridge National Laboratory.

  9. REGIONAL CONFERENCE SUMMARIES, 1966.

    ERIC Educational Resources Information Center

    Bureau of Adult, Vocational, and Technical Education (DHEW/OE), Washington, DC. Div. of Vocational and Technical Education.

    AN AVERAGE OF 200 TEACHER EDUCATORS, STATE DIRECTORS, LAYMEN, AND REPRESENTATIVES OF VARIOUS AGENCIES ATTENDED EACH OF NINE REGIONAL CONFERENCES CONDUCTED THROUGHOUT THE UNITED STATES TO DISCUSS THE INFLUENCE OF SOCIAL AND ECONOMIC CHANGES AND PROBLEMS IN PLANNING AND CONDUCTING VOCATIONAL AND TECHNICAL EDUCATION PROGRAMS. MAJOR SPEECHES PRESENTED…

  10. Recipe for Regional Development.

    ERIC Educational Resources Information Center

    Baldwin, Fred D.

    1994-01-01

    The Ceramics Corridor has created new jobs in New York's Appalachian region by fostering ceramics research and product development by small private companies. Corridor business incubators offer tenants low overhead costs, fiber-optic connections to Alfred University's mainframe computer, rental of lab space, and use of equipment small companies…

  11. Multiethnic Societies and Regions.

    ERIC Educational Resources Information Center

    Stanfield, John H., II

    1996-01-01

    Maintains that sociology must reconceptualize the meaning of multiethnic societies and regions and also advance theories about how such social organizations came into being and transform themselves through conflicting and peaceful processes. Briefly reviews traditional approaches and outlines new areas of study. (MJP)

  12. Regionalism. Clip and Save.

    ERIC Educational Resources Information Center

    Hubbard, Guy

    2002-01-01

    Focuses on the art movement, called Regionalism, discussing the painters involved and describing the characteristics of the art movement. Provides a set of learning activities and background information on John Steuart Curry. Includes a discussion of Curry's painting, "Tornado Over Kansas," and a reproduction of the painting. (CMK)

  13. MISR Regional SAMUM Products

    Atmospheric Science Data Center

    2016-08-24

    ... Regional products:  Radiance ,  Aerosol , and  Land Surface . Each product summarizes selected parameters from one Level 1 or ... the  MISR SAMUM data table . Images available on this web site include the following parameters: Image Description ...

  14. MISR Regional VBBE Products

    Atmospheric Science Data Center

    2016-08-24

    ... Regional products:  Radiance ,  Aerosol , and  Land Surface . Each product summarizes selected parameters from one Level 1 or ... the  MISR VBBE data table . Images available on this web site include the following parameters: Image Description ...

  15. Climatic Concepts and Regions.

    ERIC Educational Resources Information Center

    Thomas, Paul F.

    Designed for students in grades 7 through 12, this teaching unit presents illustrative resource materials depicting concepts related to climate and geographic regions. Emphasis is on giving students an understanding of climatic elements and factors, not as isolated, disjointed entities, but as a dynamic interplay of forces having a very definite…

  16. Australia's Regional Youth Exodus.

    ERIC Educational Resources Information Center

    Gabriel, M.

    2002-01-01

    Examines media coverage of youth outmigration from Tasmania in the context of Australia's regional crisis. Focuses on how young people are constructed by others and positioned in others' visions of their rural home towns. Discusses two recurring narratives: strategies to keep youth at home, and preoccupation with the "best and brightest" young…

  17. Benchmarks: WICHE Region 2012

    ERIC Educational Resources Information Center

    Western Interstate Commission for Higher Education, 2013

    2013-01-01

    Benchmarks: WICHE Region 2012 presents information on the West's progress in improving access to, success in, and financing of higher education. The information is updated annually to monitor change over time and encourage its use as a tool for informed discussion in policy and education communities. To establish a general context for the…

  18. Systematic screening for mutations in the promoter and the coding region of the 5-HT{sub 1A} gene

    SciTech Connect

    Erdmann, J.; Shimron-Abarbanell, D.; Cichon, S.

    1995-10-09

    In the present study we sought to identify genetic variation in the 5-HT{sub 1A} receptor gene which through alteration of protein function or level of expression might contribute to the genetic predisposition to neuropsychiatric diseases. Genomic DNA samples from 159 unrelated subjects (including 45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette`s syndrome, as well as 25 healthy controls) were investigated by single-strand conformation analysis. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as the 5{prime} untranslated region of the 5-HT{sub 1A} gene. The region upstream to the coding sequence we investigated contains a functional promoter. We found two rare nucleotide sequence variants. Both mutations are located in the coding region of the gene: a coding mutation (A{yields}G) in nucleotide position 82 which leads to an amino acid exchange (Ile{yields}Val) in position 28 of the receptor protein and a silent mutation (C{yields}T) in nucleotide position 549. The occurrence of the Ile-28-Val substitution was studied in an extended sample of patients (n = 352) and controls (n = 210) but was found in similar frequencies in all groups. Thus, this mutation is unlikely to play a significant role in the genetic predisposition to the diseases investigated. In conclusion, our study does not provide evidence that the 5-HT{sub 1A} gene plays either a major or a minor role in the genetic predisposition to schizophrenia, bipolar affective disorder, or Tourette`s syndrome. 29 refs., 4 figs., 1 tab.

  19. Definition of regulatory sequence elements in the promoter region and the first intron of the myotonic dystrophy protein kinase gene.

    PubMed

    Storbeck, C J; Sabourin, L A; Waring, J D; Korneluk, R G

    1998-04-10

    Myotonic dystrophy is the most common inherited adult neuromuscular disorder with a global frequency of 1/8000. The genetic defect is an expanding CTG trinucleotide repeat in the 3'-untranslated region of the myotonic dystrophy protein kinase gene. We present the in vitro characterization of cis regulatory elements controlling transcription of the myotonic dystrophy protein kinase gene in myoblasts and fibroblasts. The region 5' to the initiating ATG contains no consensus TATA or CCAAT box. We have mapped two transcriptional start sites by primer extension. Deletion constructs from this region fused to the bacterial chloramphenicol acetyltransferase reporter gene revealed only subtle muscle specific cis elements. The strongest promoter activity mapped to a 189-base pair fragment. This sequence contains a conserved GC box to which the transcription factor Sp1 binds. Reporter gene constructs containing a 2-kilobase pair first intron fragment of the myotonic dystrophy protein kinase gene enhances reporter activity up to 6-fold in the human rhabdomyosarcoma myoblast cell line TE32 but not in NIH 3T3 fibroblasts. Co-transfection of a MyoD expression vector with reporter constructs containing the first intron into 10 T1/2 fibroblasts resulted in a 10-20-fold enhancement of expression. Deletion analysis of four E-box elements within the first intron reveal that these elements contribute to enhancer activity similarly in TE32 myoblasts and 10 T1/2 fibroblasts. These data suggest that E-boxes within the myotonic dystrophy protein kinase first intron mediate interactions with upstream promoter elements to up-regulate transcription of this gene in myoblasts.

  20. Isolation of cosmid and cDNA clones in the region surrounding the BTK gene at Xq21.3-q22

    SciTech Connect

    Vorechovsky, I.; Zhou, J.N.; Hammarstroem, L.

    1994-06-01

    A regional physical and transcription map involving yeast artificial chromosomes (YACs), cosmids, and cDNAs has been constructed for Xq21.3-q22 around the gene BTK (formerly atk or BPK) defective in X-linked agammaglobulinemia (XLA). With a positional cloning strategy employing direct cDNA selection, novel cDNAs were found to cluster in the region of approximately 100 kb flanking the XLA and {alpha}-galactosidase A loci. While these widely expressed transcripts are in the area known to contain CpG islands, a less evolutionarily conserved gene, located more than 130 kb distal of DXS178, maps to cosmid clones that could not be digested with rare-cutting restriction enzymes. The presence of transcribed sequences flanking the BTK allowed investigation of their involvement in complex XLA phenotypes. Southern blot analysis using cDNA clones isolated from this region permitted exclusion of a contiguous deletion syndrome as an underlying defect in three patients with XLA and associated growth hormone deficiency. A single XLA patient with torsion dystonia and cosegregating X-linked deafness has been found with a deletion in the 3{prime} part of BTK extending centromerically into the flanking expressed sequence DXS1274E. This suggests a possible involvement of the DXS1274E in this phenotype. The GenBank accession numbers for novel cDNA sequences are as follows: DXS1269E (L20773), DXS1271E (UO1923), DXS1273E (UO1925), and DXS1274E (UO1922). 51 refs., 4 figs., 1 tab.

  1. Structure, sequence, expression, and chromosomal localization of the human V{sub 1a} vasopressin receptor gene

    SciTech Connect

    Thibonnier, M.; Graves, M.K.; Wagner, M.S.

    1996-02-01

    We recently reported the structure and functional expression of a human V{sub 1a} vasopressin receptor (V{sub 1a}R) cDNA isolated from human liver cDNA libraries. To understand further the expression and regulation of the V{sub 1a}R, we now describe the genomic characteristics, tissue expression, chromosomal localization, and regional mapping of the human V{sub 1a}R gene, AVPR1A. Tissue distribution of the human V{sub 1a}R mRNA explored by Northern blot analysis of various human tissues or organs revealed the presence of a 5.5-kb mRNA transcript expressed in the liver and to a lesser degree in the heart, the kidney, and skeletal muscle. Screening of human genomic libraries revealed that the human AVPR1A gene is included entirely within a 6.4-kb rated by a 2.2-kb intron located before the corresponding seventh transmembrane domain of the receptor sequence. The first exon also contains 2 kb of 5{prime}-untranslated region, and the second exon includes 1 kb of 3{prime}-untranslated region. 5{prime}-RACE analysis of human liver mRNA by PCR localized the V{sub 1a}R mRNA transcription start site 1973 bp upstream of the translation the intron sequence were used as primers in polymerase chain reaction (PCR) analysis of human/rodent somatic cell hybrids. AVPR1A was localized by PCR analysis of a somatic cell hybrid panel to chromosome 12. Fluorescence in situ hybridization using a yeast artificial chromosome physically mapped AVPR1A to region 12q14-q15. 34 refs., 4 figs.

  2. [Region 1112-1123 in the central domain of 18S rRNA in 40S subunits of plant ribosomes: accessibility for complementary interactions and the functional role].

    PubMed

    Zhigaĭlov, A V; Graĭfer, D M; Babaĭlova, E S; Polimbetova, N S; Karpova, G G; Iskakov, B K

    2010-01-01

    The binding of the 18S RNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112-1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5'-untranslated region upstream of the reporter sequence coding for beta-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5' end allowed for the demonstration that the 1112-1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112-1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.

  3. Statistical region merging.

    PubMed

    Nock, Richard; Nielsen, Frank

    2004-11-01

    This paper explores a statistical basis for a process often described in computer vision: image segmentation by region merging following a particular order in the choice of regions. We exhibit a particular blend of algorithmics and statistics whose segmentation error is, as we show, limited from both the qualitative and quantitative standpoints. This approach can be efficiently approximated in linear time/space, leading to a fast segmentation algorithm tailored to processing images described using most common numerical pixel attribute spaces. The conceptual simplicity of the approach makes it simple to modify and cope with hard noise corruption, handle occlusion, authorize the control of the segmentation scale, and process unconventional data such as spherical images. Experiments on gray-level and color images, obtained with a short readily available C-code, display the quality of the segmentations obtained.

  4. Active region seismology

    NASA Technical Reports Server (NTRS)

    Bogdan, Tom; Braun, D. C.

    1995-01-01

    Active region seismology is concerned with the determination and interpretation of the interaction of the solar acoustic oscillations with near-surface target structures, such as magnetic flux concentration, sunspots, and plage. Recent observations made with a high spatial resolution and a long temporal duration enabled measurements of the scattering matrix for sunspots and solar active regions to be carried out as a function of the mode properties. Based on this information, the amount of p-mode absorption, partial-wave phase shift, and mode mixing introduced by the sunspot, could be determined. In addition, the possibility of detecting the presence of completely submerged magnetic fields was raised, and new procedures for performing acoustic holography of the solar interior are being developed. The accumulating evidence points to the mode conversion of p-modes to various magneto-atmospheric waves within the magnetic flux concentration as being the unifying physical mechanism responsible for these diverse phenomena.

  5. Arctic region mapping tool

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2012-08-01

    An interactive online mapping tool is now available to assist with scientific, environmental, and emergency response needs in the Arctic region, the National Oceanic and Atmospheric Administration (NOAA) announced on 31 July. The Environmental Response Management Application (ERMA®) already has been used in other regions, including in the Gulf of Mexico, as part of the response to the Deepwater Horizon oil spill in 2010. The tool—which is a product of the combined work of NOAA, the U.S. Department of the Interior's Bureau of Safety and Environmental Enforcement (BSEE), the University of New Hampshire, and others—offers near-real time oceanographic observations, weather data, environmental and commercial information, and other data.

  6. Regional Renewable Energy Cooperatives

    NASA Astrophysics Data System (ADS)

    Hazendonk, P.; Brown, M. B.; Byrne, J. M.; Harrison, T.; Mueller, R.; Peacock, K.; Usher, J.; Yalamova, R.; Kroebel, R.; Larsen, J.; McNaughton, R.

    2014-12-01

    We are building a multidisciplinary research program linking researchers in agriculture, business, earth science, engineering, humanities and social science. Our goal is to match renewable energy supply and reformed energy demands. The program will be focused on (i) understanding and modifying energy demand, (ii) design and implementation of diverse renewable energy networks. Geomatics technology will be used to map existing energy and waste flows on a neighbourhood, municipal, and regional level. Optimal sites and combinations of sites for solar and wind electrical generation (ridges, rooftops, valley walls) will be identified. Geomatics based site and grid analyses will identify best locations for energy production based on efficient production and connectivity to regional grids and transportation. Design of networks for utilization of waste streams of heat, water, animal and human waste for energy production will be investigated. Agriculture, cities and industry produce many waste streams that are not well utilized. Therefore, establishing a renewable energy resource mapping and planning program for electrical generation, waste heat and energy recovery, biomass collection, and biochar, biodiesel and syngas production is critical to regional energy optimization. Electrical storage and demand management are two priorities that will be investigated. Regional scale cooperatives may use electric vehicle batteries and innovations such as pump storage and concentrated solar molten salt heat storage for steam turbine electrical generation. Energy demand management is poorly explored in Canada and elsewhere - our homes and businesses operate on an unrestricted demand. Simple monitoring and energy demand-ranking software can easily reduce peaks demands and move lower ranked uses to non-peak periods, thereby reducing the grid size needed to meet peak demands. Peak demand strains the current energy grid capacity and often requires demand balancing projects and

  7. Northwest Regional Climate Assessment

    NASA Technical Reports Server (NTRS)

    Lipschultz, Fred

    2011-01-01

    Objectives are to establish a continuing, inclusive National process that: 1) synthesizes relevant science and information 2) increases understanding of what is known & not known 3) identifies information needs related to preparing for climate variability and change, and reducing climate impacts and vulnerability 4) evaluates progress of adaptation & mitigation activities 5) informs science priorities 6) builds assessment capacity in regions and sectors 7) builds understanding & skilled use of findings

  8. Complex regional pain syndrome.

    PubMed

    Bruehl, Stephen

    2015-07-29

    Complex regional pain syndrome is a chronic pain condition characterized by autonomic and inflammatory features. It occurs acutely in about 7% of patients who have limb fractures, limb surgery, or other injuries. Many cases resolve within the first year, with a smaller subset progressing to the chronic form. This transition is often paralleled by a change from "warm complex regional pain syndrome," with inflammatory characteristics dominant, to "cold complex regional pain syndrome" in which autonomic features dominate. Multiple peripheral and central mechanisms seem to be involved, the relative contributions of which may differ between individuals and over time. Possible contributors include peripheral and central sensitization, autonomic changes and sympatho-afferent coupling, inflammatory and immune alterations, brain changes, and genetic and psychological factors. The syndrome is diagnosed purely on the basis of clinical signs and symptoms. Effective management of the chronic form of the syndrome is often challenging. Few high quality randomized controlled trials are available to support the efficacy of the most commonly used interventions. Reviews of available randomized trials suggest that physical and occupational therapy (including graded motor imagery and mirror therapy), bisphosphonates, calcitonin, subanesthetic intravenous ketamine, free radical scavengers, oral corticosteroids, and spinal cord stimulation may be effective treatments. Multidisciplinary clinical care, which centers around functionally focused therapies is recommended. Other interventions are used to facilitate engagement in functional therapies and to improve quality of life.

  9. Regional Shelter Analysis Methodology

    SciTech Connect

    Dillon, Michael B.; Dennison, Deborah; Kane, Jave; Walker, Hoyt; Miller, Paul

    2015-08-01

    The fallout from a nuclear explosion has the potential to injure or kill 100,000 or more people through exposure to external gamma (fallout) radiation. Existing buildings can reduce radiation exposure by placing material between fallout particles and exposed people. Lawrence Livermore National Laboratory was tasked with developing an operationally feasible methodology that could improve fallout casualty estimates. The methodology, called a Regional Shelter Analysis, combines the fallout protection that existing buildings provide civilian populations with the distribution of people in various locations. The Regional Shelter Analysis method allows the consideration of (a) multiple building types and locations within buildings, (b) country specific estimates, (c) population posture (e.g., unwarned vs. minimally warned), and (d) the time of day (e.g., night vs. day). The protection estimates can be combined with fallout predictions (or measurements) to (a) provide a more accurate assessment of exposure and injury and (b) evaluate the effectiveness of various casualty mitigation strategies. This report describes the Regional Shelter Analysis methodology, highlights key operational aspects (including demonstrating that the methodology is compatible with current tools), illustrates how to implement the methodology, and provides suggestions for future work.

  10. The Pacific Region.

    PubMed

    Tagica, K

    1993-03-01

    Population education in the Pacific region is summarized in terms of awareness and commitment, curriculum and instructional materials development, integration into the school curricula, training programs, and evaluation research. Several population education issues of current concern relate to the increase in chronic diseases such as diabetes and hypertension that are associated with life styles and diet, and the rising incidence of AIDS and teenage pregnancy. In the Pacific region, many countries have advanced population programs and policies, while some still do not even have a population policy. The issue of balancing population and resources is a topic that has not been sufficiently addressed in resource-poor countries. There is wide variance in awareness and commitment to population education in the Pacific region. Commitment and continuous support are crucial to population education projects. Lack of support is sometimes due to changing government personnel and lack of awareness of policy makers. Population education is not the same as family planning or sex education, and traditionally is spread through seminars and workshops by part time project personnel unconnected to the entire educational apparatus. Presently, only 8 population projects are functioning in the region, with 2-3 in the planning stages. Materials development in the Pacific region has been devoted to the secondary school level, yet awareness is increasing that sexuality, family health, and the environment should be introduced at the primary level. A popular strategy is to integrate population issues into the existing curriculum, such as in Fiji, the Marshall Islands, and Kiribati, which also have teacher training curriculum. In most countries sex education is still a controversial topic, and materials are developed by teacher committees working after school rather in a curriculum development unit. AIDS has pushed this topic into the public sector. A chart is provided for each country and

  11. Venus - Eistla Region

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This Magellan image is of an area located in the Eistla Region of Venus in the southern hemisphere and is centered at 5.5 degrees east longitude, 18 degrees south latitude. It is 122 kilometers (76 miles) across east to west and 107 kilometers (66 miles) north to south. North is at the top of the image. Shown is an unusual volcanic edifice unlike all others previously observed. It is approximately 66 kilometers (41 miles) across at the base and has a relatively flat, slightly concave summit 35 kilometers (22 miles) in diameter. The sides of the edifice are characterized by radiating ridges and valleys that impart a fluted appearance. To the west, the rim of the structure appears to have been breached by dark lava flows that emanated from a shallow summit pit approximately 5 kilometers (3 miles) in diameter and traveled west along a channel approximately 5 kilometers wide and 27 kilometers (17 miles) long. A series of coalescing, collapsed pits 2 to 10 kilometers (1.2 to 6.2 miles) in diameter are located 10 kilometers (6 miles) west of the summit. The edifice and western pits are circumscribed by faint, concentric lineaments up to 70 kilometers (43 miles) in diameter. A series of north northwest trending graben are deflected eastward around the edifice; the interplay of these graben and the fluted rim of the edifice produce a distinctive scalloped pattern in the image. Several north northwest trending lineaments cut directly across the summit region. This peculiar volcanic construct is located 25 to 30 kilometers (15 to 19 miles) north of Alpha Regio, a highly deformed region of tessera terrain. A collection of at least six similar volcanoes has been observed near Thetis Regio, a region of tessera within Aphrodite Terra. Thus, these unusual constructs tentatively appear to be spatially associated with regions of tessera. A tessera is a complex, deformed terrain on Venus consisting of at least two sets of intersecting ridges and troughs. The implications of this

  12. Cloning and functional characterization of the 5' regulatory region of ovine Hormone Sensitive Lipase (HSL) gene.

    PubMed

    Lampidonis, Antonis D; Stravopodis, Dimitrios J; Voutsinas, Gerassimos E; Messini-Nikolaki, Niki; Stefos, George C; Margaritis, Lukas H; Argyrokastritis, Alexandros; Bizelis, Iosif; Rogdakis, Emmanuel

    2008-12-31

    Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL (ovHSL) gene transcription activity, we initially isolated and cloned the 5' proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5'-untranslated region (5'-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5' to 3') cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and

  13. Regional river sulfur runoff

    NASA Astrophysics Data System (ADS)

    Husar, Rudolf B.; Husar, Janja Djukic

    1985-01-01

    The water and sulfur runoff data for 54 large river basins were assembled, covering 65% of the nondesert land area of the world. The sulfur concentration ranges from 0.5 mg S/L for the West African rivers Niger and Volta to 100 mg S/L in the Colorado River; the world average is 3.2 mg S/L. The concentrations in central and eastern Europe as well as central and eastern North America exceed 8 mg S/L. The sulfur runoff density is also highest in the river basins over these industrialized regions, exceeding 2 g S/m2/yr. However, high sulfur runoff density in excess of 3 g S/m2/yr is also measured over the Pacific islands New Zealand and New Guinea and the archipelagos of Indonesia and the Philippines. The natural background sulfur runoff was estimated by assuming that South America, Africa, Australia, and the Pacific Islands are unperturbed by man and that the average river sulfur concentration is in the range 1-3 mg S/L. Taking these background concentration values, the man-induced sulfur runoff for Europe ranges between 2 and 8 times the natural flow, and over North America, man's contribution ranges between 1 and 5 times the natural runoff. The global sulfur flow from nondesert land to the oceans and the Caspian Sea is estimated as 131 Tg S/yr, of which 46-85 Tg S/yr is attributed to natural causes. The regional river sulfur runoff pattern discussed in this paper does not have enough spatial resolution to be directly applicable to studies of the environmental effects of man-induced sulfur flows. However, it points to the continental-size regions where those perturbations are most evident and to the magnitude of the perturbations as expressed in units of the natural flows.

  14. Regional river sulfur runoff

    SciTech Connect

    Husar, R.B.; Husar, J.D.

    1985-01-20

    The water and sulfur runoff data for 54 large river basins were assembled, covering 65% of the nondesert land area of the world. The sulfur concentration ranges from 0.5 mg S/L for the West African rivers Niger and Volta to 100 mg S/L in the Colorado River; the world average is 3.2 mg S/L. The concentrations in central and eastern Europe as well as central and eastern North America exceed 8 mg S/L. The sulfur runoff density is also highest in the river basins over these industrialized regions, exceeding 2 g S/m/sup 2//yr. However, high sulfur runoff density in excess of 3 g S/m/sup 2//yr is also measured over the Pacific islands New Zealand and New Guinea and the archipelagos of Indonesia and the Philippines. The natural background sulfur runoff was estimated by assuming that South America, Africa, Australia, and the Pacific Islands are unperturbed by man and that the average river sulfur concentration is in the range 1--3 mg S/L. Taking these background concentration values, the man-induced sulfur runoff for Europe ranges between 2 and 8 times the natural flow, and over North America, man's contribution ranges between 1 and 5 times the natural runoff. The global sulfur flow from nondesert land to the oceans and the Caspian Sea is estimated as 131 Tg S/yr, of which 46--85 Tg S/yr is attributed to natural causes. The regional river sulfur runoff pattern discussed in this paper does not have enough spatial resolution to be directly applicable to studies of the environmental effects of man-induced sulfur flows. However, it points to the continental-size regions where those perturbations are most evident and to the magnitude of the perturbations as expressed in units of the natural flows.

  15. Higher Education and Regional Development.

    ERIC Educational Resources Information Center

    Neave, Guy

    1979-01-01

    The effect that the university has upon its region and the issue of regional control of higher education are examined. A definition of regional development is offered and regional planning, relevant research, cultural mobilization, and the Jacobin university are described. (Author/MLW)

  16. Complex regional pain syndrome.

    PubMed

    Sebastin, Sandeep J

    2011-05-01

    Complex regional pain syndrome (CRPS) previously known as reflex sympathetic dystrophy is a chronic neurological disorder involving the limbs characterized by disabling pain, swelling, vasomotor instability, sudomotor abnormality, and impairment of motor function. CRPS is not uncommon after hand surgery and may complicate post-operative care. There is no specific diagnostic test for CRPS and the diagnosis is based on history, clinical examination, and supportive laboratory findings. Recent modifications to diagnostic criteria have enabled clinicians to diagnose this disease more consistently. This review gives a synopsis of CRPS and discusses the diagnosis, pathophysiology, and treatment options based on the limited evidence in the literature. PMID:22022040

  17. Complex regional pain syndrome

    PubMed Central

    Sebastin, Sandeep J

    2011-01-01

    Complex regional pain syndrome (CRPS) previously known as reflex sympathetic dystrophy is a chronic neurological disorder involving the limbs characterized by disabling pain, swelling, vasomotor instability, sudomotor abnormality, and impairment of motor function. CRPS is not uncommon after hand surgery and may complicate post-operative care. There is no specific diagnostic test for CRPS and the diagnosis is based on history, clinical examination, and supportive laboratory findings. Recent modifications to diagnostic criteria have enabled clinicians to diagnose this disease more consistently. This review gives a synopsis of CRPS and discusses the diagnosis, pathophysiology, and treatment options based on the limited evidence in the literature. PMID:22022040

  18. Complex regional pain syndrome.

    PubMed

    Palmer, Greta

    2015-06-01

    Complex regional pain syndrome is an uncommon chronic pain condition. It develops spontaneously or following an injury. The features are limb pain, allodynia, hypersensitivity, hyperalgesia, abnormalities of the vasomotor, sudomotor and motor systems, and trophic changes, with reduced use of the affected limb. The diagnosis is clinical and one of exclusion. The emphasis of therapy is graded rehabilitation and movement of the limb with physiotherapy and occupational therapy. Psychological therapies should be offered if a patient is making no or slow progress in the acute phase, and to all patients in the chronic phase as depression can occur. The goal of pharmacotherapy is to assist functional improvement. The early phase may be managed with simple analgesia. Antineuropathic drugs including tricyclic antidepressants and antiepileptic drugs may be added. Other treatments with some evidence of effectiveness include corticosteroids, calcitonin and bisphosphonates. Vitamin C has been used for primary prevention after wrist fracture and upper and lower limb surgery. There is no evidence that it is effective for treating established complex regional pain syndrome.

  19. Colorado Regional Faults

    SciTech Connect

    Hussein, Khalid

    2012-02-01

    Citation Information: Originator: Earth Science &Observation Center (ESOC), CIRES, University of Colorado at Boulder Originator: Colorado Geological Survey (CGS) Publication Date: 2012 Title: Regional Faults Edition: First Publication Information: Publication Place: Earth Science & Observation Center, Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder Publisher: Earth Science &Observation Center (ESOC), CIRES, University of Colorado at Boulder Description: This layer contains the regional faults of Colorado Spatial Domain: Extent: Top: 4543192.100000 m Left: 144385.020000 m Right: 754585.020000 m Bottom: 4094592.100000 m Contact Information: Contact Organization: Earth Science &Observation Center (ESOC), CIRES, University of Colorado at Boulder Contact Person: Khalid Hussein Address: CIRES, Ekeley Building Earth Science & Observation Center (ESOC) 216 UCB City: Boulder State: CO Postal Code: 80309-0216 Country: USA Contact Telephone: 303-492-6782 Spatial Reference Information: Coordinate System: Universal Transverse Mercator (UTM) WGS’1984 Zone 13N False Easting: 500000.00000000 False Northing: 0.00000000 Central Meridian: -105.00000000 Scale Factor: 0.99960000 Latitude of Origin: 0.00000000 Linear Unit: Meter Datum: World Geodetic System 1984 (WGS ’984) Prime Meridian: Greenwich Angular Unit: Degree Digital Form: Format Name: Shape file

  20. Regional flood probabilities

    USGS Publications Warehouse

    Troutman, B.M.; Karlinger, M.R.

    2003-01-01

    The T-year annual maximum flood at a site is defined to be that streamflow, that has probability 1/T of being exceeded in any given year, and for a group of sites the corresponding regional flood probability (RFP) is the probability that at least one site will experience a T-year flood in any given year. The RFP depends on the number of sites of interest and on the spatial correlation of flows among the sites. We present a Monte Carlo method for obtaining the RFP and demonstrate that spatial correlation estimates used in this method may be obtained with rank transformed data and therefore that knowledge of the at-site peak flow distribution is not necessary. We examine the extent to which the estimates depend on specification of a parametric form for the spatial correlation function, which is known to be nonstationary for peak flows. It is shown in a simulation study that use of a stationary correlation function to compute RFPs yields satisfactory estimates for certain nonstationary processes. Application of asymptotic extreme value theory is examined, and a methodology for separating channel network and rainfall effects on RFPs is suggested. A case study is presented using peak flow data from the state of Washington. For 193 sites in the Puget Sound region it is estimated that a 100-year flood will occur on the average every 4,5 years.

  1. The Eutherian Pseudoautosomal Region.

    PubMed

    Raudsepp, Terje; Chowdhary, Bhanu P

    2015-01-01

    The pseudoautosomal region (PAR) is a unique segment of sequence homology between differentiated sex chromosomes where recombination occurs during meiosis. Molecular and functional properties of the PAR are distinctive from the autosomes and the remaining regions of the sex chromosomes. These include a higher rate of recombination than genome average, bias towards GC-substitutions and increased interindividual nucleotide divergence and mutations. As yet, the PAR has been physically demarcated in only 28 eutherian species representing 6 mammalian orders. Murid rodents have the smallest, gene-poorest and most diverged PARs. Other eutherian PARs are largely homologous but differ in size and gene content, being the smallest in equids and human/simian primates and much larger in other eutherians. Because pseudoautosomal genes escape X inactivation, their dosage changes with sex chromosome aneuploidies, whereas phenotypic effects of the latter depend on the size and gene content of the PAR. Thus, X monosomy is more viable in mice, humans and horses than in species with larger PARs. Presently, little is known about the functions of PAR genes in individual species, though human studies suggest their involvement in early embryonic development. The PAR is, thus, of evolutionary, genetic and biomedical significance and a 'research hotspot' in eutherian genomes. PMID:26730606

  2. Complex regional pain syndrome.

    PubMed

    Palmer, Greta

    2015-06-01

    Complex regional pain syndrome is an uncommon chronic pain condition. It develops spontaneously or following an injury. The features are limb pain, allodynia, hypersensitivity, hyperalgesia, abnormalities of the vasomotor, sudomotor and motor systems, and trophic changes, with reduced use of the affected limb. The diagnosis is clinical and one of exclusion. The emphasis of therapy is graded rehabilitation and movement of the limb with physiotherapy and occupational therapy. Psychological therapies should be offered if a patient is making no or slow progress in the acute phase, and to all patients in the chronic phase as depression can occur. The goal of pharmacotherapy is to assist functional improvement. The early phase may be managed with simple analgesia. Antineuropathic drugs including tricyclic antidepressants and antiepileptic drugs may be added. Other treatments with some evidence of effectiveness include corticosteroids, calcitonin and bisphosphonates. Vitamin C has been used for primary prevention after wrist fracture and upper and lower limb surgery. There is no evidence that it is effective for treating established complex regional pain syndrome. PMID:26648626

  3. Neptune's south polar region

    NASA Technical Reports Server (NTRS)

    1989-01-01

    This image of Neptune's south polar region was obtained by the NASA Voyager narrow-angle camera on Aug. 23, 1989, when it was at a distance of 25 million kilometers (1.6 million miles). The smallest cloud features are 45 kilometers (28 miles) in diameter. The image shows the discovery of shadows in Neptune's atmosphere, shadows cast onto a deep cloud bank by small elevated clouds. Located at about 68 degrees south latitude, they are the first cloud shadows ever seen by the Voyager on any planet. The dark regions adjacent to the small bright clouds are believed to be shadows, because they are on the side of the cloud that is opposite to the incoming sunlight and because they lengthen in places where the sun lies closer to the horizon. Estimates of the height of these discrete clouds above the underlying cloud bank can be obtained by careful analysis of this data. The Voyager Mission is conducted by JPL for NASA's Office of Space Science and Applications.

  4. Characterization and genomic mapping of genes and pseudogenes of a new human protein tyrosine phosphatase

    SciTech Connect

    Zhao, Zhaoyang; Lee, Cheng-Chi; Monckton, D.G.

    1996-07-01

    Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features. Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A). The 167-amino-acid human PTPs, but does not show any other sequence homology to any of the previously described PTPs. Two cDNA encoding PTP4A that differed in their noncoding regions were isolated. Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated. Northern analysis using a probe from a common 3{prime}-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb. Both species of mRNA were seen in all human adult and fetal tissues tested. Fluorescence in situ hybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11. A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs. Our studies also suggest the existence of another processed pseudogene on chromosome 11. 31 refs., 6 figs., 1 tab.

  5. Identification and characterization of Kaposi's sarcoma-associated herpesvirus open reading frame 11 promotor activation

    SciTech Connect

    Chen, Lei

    2008-01-01

    Open reading frame 11 (ORF11) of Kaposi's sarcoma-associated herpesvirus belongs to a herpesviral homologous protein family shared by some members of the gamma- herpesvirus subfamily. Little is known about this ORF11 homologous protein family. We have characterized an unknown open reading frame, ORF11, located adjacent and in the opposite orientation to a well-characterized viral IL-6 gene. Northern blot analysis reveals that ORF11 is expressed during the KSHV lytic cycle with delayed-early transcription kinetics. We have determined the 5{prime} and 3{prime} untranslated region of the unspliced ORF11 transcript and identified both the transcription start site and the transcription termination site. Core promoter region, representing ORF11 promoter activity, was mapped to a 159nt fragment 5{prime} most proximal to the transcription start site. A functional TATA box was identified in the core promoter region. Interestingly, we found that ORF11 transcriptional activation is not responsive to Rta, the KSHV lytic switch protein. We also discovered that part of the ORF11 promoter region, the 209nt fragment upstream of the transcription start site, was repressed by phorbol esters. Our data help to understand transcription regulation of ORF11 and to elucidate roles of ORF11 in KSHV pathogenesis and life cycle.

  6. [COMPLEX REGIONAL PAIN SYNDROME].

    PubMed

    Blažeković, Ivan; Bilić, Ervina; Žagar, Marija; Anić, Branimir

    2015-01-01

    Complex regional pain syndrome (CRPS) represents a state of constant and often disabling pain, affecting one region (usually hand) and often occurs after a trauma whose severity does not correlate with the level of pain. The older term for this condition of chronic pain associated with motor and autonomic symptoms is reflex sympathetic dystrophy or causalgia. The aim of this review, based on contemporary literature, is to show the epidemiology and etiology, proposed pathophysiological mechanisms, method of diagnosis and treatment options, prevention and mitigation of this under-recognized disease. CRPS I occurs without known neurological damage, unlike CRPS II, where the history of trauma is present and in some cases damage to the peripheral nervous system can be objectively assessed using electromyoneurography. New diagnostic methods, such as quantitative sensory testing (CST), challenge this division because the CST findings in patients with CRPS I can suggest damage to Adelta peripheral nerve fibers. Except for distinguishing type I and type II disease, it is important to bear in mind the diversity of clinical presentation of CRPS in acute and chronic phase of the disease. This regional pain syndrome typically includes the autonomic and motor signs and thus differs from other peripheral neuropathic pain syndromes. The complexity of the clinical presentation indicates the likely presence of different pathophysiological mechanisms underlying this disease. Previous studies have demonstrated the autonomic dysfunction, neurogenic inflammation and neuroplastic changes. The diagnosis of CRPS is based on anamnesis and clinical examination on the basis of which the disease can be graded according to the Budapest Criteria. A valuable aid in differentiating subtypes of the disease is electromyoneurography. The treatment of CRPS is as complex as the clinical picture and the pathophysiology of the disease and requires interdisciplinary cooperation and individual approach

  7. [COMPLEX REGIONAL PAIN SYNDROME].

    PubMed

    Blažeković, Ivan; Bilić, Ervina; Žagar, Marija; Anić, Branimir

    2015-01-01

    Complex regional pain syndrome (CRPS) represents a state of constant and often disabling pain, affecting one region (usually hand) and often occurs after a trauma whose severity does not correlate with the level of pain. The older term for this condition of chronic pain associated with motor and autonomic symptoms is reflex sympathetic dystrophy or causalgia. The aim of this review, based on contemporary literature, is to show the epidemiology and etiology, proposed pathophysiological mechanisms, method of diagnosis and treatment options, prevention and mitigation of this under-recognized disease. CRPS I occurs without known neurological damage, unlike CRPS II, where the history of trauma is present and in some cases damage to the peripheral nervous system can be objectively assessed using electromyoneurography. New diagnostic methods, such as quantitative sensory testing (CST), challenge this division because the CST findings in patients with CRPS I can suggest damage to Adelta peripheral nerve fibers. Except for distinguishing type I and type II disease, it is important to bear in mind the diversity of clinical presentation of CRPS in acute and chronic phase of the disease. This regional pain syndrome typically includes the autonomic and motor signs and thus differs from other peripheral neuropathic pain syndromes. The complexity of the clinical presentation indicates the likely presence of different pathophysiological mechanisms underlying this disease. Previous studies have demonstrated the autonomic dysfunction, neurogenic inflammation and neuroplastic changes. The diagnosis of CRPS is based on anamnesis and clinical examination on the basis of which the disease can be graded according to the Budapest Criteria. A valuable aid in differentiating subtypes of the disease is electromyoneurography. The treatment of CRPS is as complex as the clinical picture and the pathophysiology of the disease and requires interdisciplinary cooperation and individual approach

  8. The high-affinity interleukin 8 receptor gene (IL8RA) maps to the 2q33-q36 region of the human genome: Cloning of a pseudogene (IL8RBP) for the low-affinity receptor

    SciTech Connect

    Mollereau, C. Laboratoire de Pharmacologie et Toxicologie Fondamentale du CNRS, Toulouse ); Muscatelli, F.; Mattei, M.G. ); Vassart, G. Universite libre de Bruxelles ); Parmentier, M. )

    1993-04-01

    The selective amplification by polymerase chain reaction (PCR) of gene fragments corresponding to new G-protein-coupled receptors resulted in the cloning of 18 orphan members of this gene family. Of these, three human clones amplified from genomic DNA (HGMP03, HGMP04, and HGMP05) were shown to be structurally related. Genomic clones corresponding to HGMP03 and HGMP05 were isolated and their putative coding region sequenced. Following the characterization of two interleukin 8 (IL-8) receptors, HGMP03 appeared to encode the high-affinity IL-8 receptor, whereas the partial clone HGMP04 encodes the low-affinity IL-8 receptor. Comparison with the cDNA sequence suggests that the high-affinity receptor gene is split by an intron in the 5[prime] untranslated region. The high-affinity receptor gene was mapped by in situ hybridization to the 2q33-q36 region of the human genome. The HGMP05 locus turned out to be a pseudogene for the low-affinity IL-8 receptor (87% identity), with multiple frameshifts and point mutations introducing stop codons. Southern blotting on genomic DNA did not allow the further detection of related loci in the human genome. 12 refs., 2 figs.

  9. Complex Regional Pain Syndrome.

    PubMed

    Ersoy, Hale; Pomeranz, Stephen J

    2016-01-01

    Complex regional pain syndrome (CRPS) is a neurological disorder producing peripheral neurogenic inflammatory process in hands and feet distal to injury, which may lead to severe disability. Symptoms are often out of proportion to the initiating event and not limited to a single peripheral nerve. There is no gold standard in diagnosis of this entity, and a multidisciplinary approach is necessary for proper diagnosis. Magnetic resonance imaging (MRI) is one of the most useful diagnostic modalities in early stages of CRPS (when clinical diagnosis is most difficult), the most desirable time to diagnose this disorder to expedite treatment and improve function. This article discusses MRI findings of CRPS, particularly in the early phase, and differential considerations. PMID:27518298

  10. Venus - Lakshmi Region

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This Magellan image is centered at 55 degrees north latitude, 348.5 degrees longitude, in the eastern Lakshmi region of Venus. This image, which is of an area 300 kilometers (180 miles) in width and 230 kilometers (138 miles) in length, is a mosaic of orbits 458 through 484. The image shows a relatively flat plains region composed of many lava flows. The dark flows mostly likely represent smooth lava flows similar to 'pahoehoe' flows on Earth while the brighter lava flows are rougher flows similar to 'aa' flows on Earth. (The terms 'pahoehoe' and 'aa' refer to textures of lava with pahoehoe a smooth or ropey surface, and aa a rough, clinkery texture). The rougher flows are brighter because the rough surface returns more energy to the radar than the smooth flows. Situated on top of the lava flows are three dark splotches. Because of the thick Venusian atmosphere, the small impactors break up before they reached the surface. Only the fragments from the broken up impactor are deposited on the surface and these fragments produce the dark splotches in this image. The splotch at the far right (east) has a crater centered in it, indicating that the impactor was not completely destroyed during its journey through the atmosphere. The dark splotches in the center and to the far left in this image each represent an impactor that was broken up into small fragments that did not penetrate the surface to produce a crater. The dark splotch at the left has been modified by the wind. A southwest northeast wind flow has moved some of the debris making up the splotch to the northeast where it has piled up against some small ridges.

  11. Moldova. Historic regional conference.

    PubMed

    Moshin, V

    1995-05-01

    The Directorate of Maternal and Child Health and the Family Planning Association of Moldova organized a regional conference, which was held October 18-19, 1994, in Kishinev, Moldova, with the support of the United Nations Population Fund (UNFPA), the World Health Organization (WHO), and the International Planned Parenthood Federation (IPPF). The conference,"Problems of Family Planning in Eastern Europe," was attended by approximately 400 Moldovan delegates of governmental and nongovernmental organizations (NGOs), and by 25 delegates from Romania, Russia, Belarus, the Ukraine, and Georgia. The President of Moldova and the Ministry of Public Health of Moldova gave their approval. The main objectives of the conference were to inform the public about the recommendations of the ICPD, to analyze the status of women's reproductive health and family planning in Eastern Europe, and to find ways of implementing the ICPD Plan of Action. Major problems identified during the conference were: 1) the social and economic problems facing most families; 2) the high rate of morbidity and mortality; 3) the decrease in birth rate; 4) the increase in abortions; 5) the rising incidence of venereal disease; and 6) the absence of an effective family planning system. It was agreed that cooperation between governments and NGOs is essential in designing population programs for each country. The following goals were set: 1) to provide populations with sufficient contraceptives; 2) to actively promote family planning concepts through the mass media; 3) to train specialists and to open family planning offices and centers; 4) to introduce sex education in the curricula of Pedagogical Institutes; and 5) to create national and regional statistical and sociological databases on population issues.

  12. The gene for human U2 snRNP auxiliary factor small 35-kDa subunit (U2AF1) maps to the progressive myoclonus epilepsy (EPM1) critical region on chromosome 21q22.3

    SciTech Connect

    Lalioti, M.D.; Rossier, C.; Antonarakis, S.E.

    1996-04-15

    We used targeted exon trapping to clone portions of genes from human chromosome 21q22.3. One trapped sequence showed complete homology with the cDNA of human U2AF{sup 35} (M96982; HGM-approved nomenclature U2AF1), which encodes for the small 35-kDa subunit of the U2 snRNP auxiliary factor. Using the U2AF1 cDNA as a probe, we mapped this gene to cosmid Q15D2, a P1, and YAC 350F7 of the Chumakov et al. contig, close to the cystathionine-{beta}-synthase gene (CBS) on 21q22.3. This localization was confirmed by PCR using oligonucleotides from the 3{prime} UTR and by FISH. As U2AF1 associated with a number of different factors during mRNA splicing, overexpression in trisomy 21 individuals could contribute to some Down syndrome phenotypes by interfering with the splicing process. Furthermore, because this gene maps in the critical region for the progressive myoclonus epilepsy I locus (EPM1), mutation analysis will be carried out in patients to evaluate the potential role of U2AF1 as a candidate for EPM1. 24 refs., 1 fig.

  13. Characterization of a human X-linked gene from the DXS732E locus in the candidate region for the anhidrotic ectodermal dysplasia (EDA) gene (Xq13.1)

    SciTech Connect

    Gault, J.; Zonana, J.; Zeltinger, J.

    1994-09-01

    A conserved mouse genomic clone was used to identify a homologous human genomic clone (the DXS732E locus), which was subsequently employed to isolate cDNAs from a human fetal brain library. Nine unique overlapping cDNAs were isolated, and sequences analysis of 3.9 kb identified a putative 1 kb ORF. GRAIL analysis of the sequence supported the hypothesis that the putative ORF was coding sequence, and Prosite analysis of the putative ORF identified potential glycosylation and phosphorylation sites. The 5{prime} end of the gene maps within a CpG island, and comparison of cDNA sequences indicate the gene is alternatively spliced at its 3{prime} end. Northern analysis and RT-PCR indicate that two different sized messages appear to be expressed with the gene expressed in human fetal kidney, intestine, brain, and muscle. The gene is expressed in 77 day human skin, a time when hair follicle formation occurs. Anhidrotic ectodermal dysplasia (EDA) results in the abnormal morphogenesis of hair, teeth and eccrine sweat glands. A positional cloning strategy towards cloning the EDA gene had been used, and deletion and X-autosome translocation patients have been useful in further delimiting the EDA region. The present gene at the DXS732E locus is partially deleted in one EDA patient who does not have other apparent abnormalities. No rearrangements of the gene have been detected in two female X-autosome translocation EDA patients, nor in four additional male patients with submicroscopic molecular deletions.

  14. Regional brain hypometabolism is unrelated to regional amyloid plaque burden.

    PubMed

    Altmann, Andre; Ng, Bernard; Landau, Susan M; Jagust, William J; Greicius, Michael D

    2015-12-01

    In its original form, the amyloid cascade hypothesis of Alzheimer's disease holds that fibrillar deposits of amyloid are an early, driving force in pathological events leading ultimately to neuronal death. Early clinicopathological investigations highlighted a number of inconsistencies leading to an updated hypothesis in which amyloid plaques give way to amyloid oligomers as the driving force in pathogenesis. Rather than focusing on the inconsistencies, amyloid imaging studies have tended to highlight the overlap between regions that show early amyloid plaque signal on positron emission tomography and that also happen to be affected early in Alzheimer's disease. Recent imaging studies investigating the regional dependency between metabolism and amyloid plaque deposition have arrived at conflicting results, with some showing regional associations and other not. We extracted multimodal neuroimaging data from the Alzheimer's disease neuroimaging database for 227 healthy controls and 434 subjects with mild cognitive impairment. We analysed regional patterns of amyloid deposition, regional glucose metabolism and regional atrophy using florbetapir ((18)F) positron emission tomography, (18)F-fluordeoxyglucose positron emission tomography and T1-weighted magnetic resonance imaging, respectively. Specifically, we derived grey matter density and standardized uptake value ratios for both positron emission tomography tracers in 404 functionally defined regions of interest. We examined the relation between regional glucose metabolism and amyloid plaques using linear models. For each region of interest, correcting for regional grey matter density, age, education and disease status, we tested the association of regional glucose metabolism with (i) cortex-wide florbetapir uptake; (ii) regional (i.e. in the same region of interest) florbetapir uptake; and (iii) regional florbetapir uptake while correcting in addition for cortex-wide florbetapir uptake. P-values for each setting

  15. Evaluation of in vitro inhibitory potential of small interfering RNAs directed against various regions of foot-and-mouth disease virus genome.

    PubMed

    Mohapatra, Jajati Keshari; Sanyal, Aniket; Hemadri, Divakar; Tosh, Chakradhar; Kumar, R Manoj; Bandyopadhyay, Santanu Kumar

    2005-04-15

    India is endemic for foot-and-mouth disease and it continues to be a major threat to the livestock industry despite vaccination programmes. In the present study, the ability of specific small interfering (si)RNAs directed against different genomic regions of foot-and-mouth disease virus (FMDV) to inhibit virus replication in BHK-21 cells was examined. For preliminary evaluation of possible siRNA-mediated FMDV inhibition, a cocktail of several unique populations of 12-30bp siRNAs were successfully produced corresponding to three target regions located at structural (VP3-VP1), non-structural (2A-2C), and non-structural-untranslated (3D-3'UTR) region of serotype Asia1. Once the populations of siRNAs generated were found to reduce the virus titre significantly, two highly conserved 21bp siRNA duplexes were designed by analysing all FMDV sequence entries available in public-domain databases. In virus titration assay, more than 99% inhibition of virus yield for all the four serotypes (type Asia1, O, A, and C) could be demonstrated in cells transfected with each of the FMDV-specific siRNAs at 24h post-infection, compared to control cells transfected with scrambled siRNA. This was well supported by reduction in OD values in FMDV-specific sandwich ELISA. Although 100-fold reduction in virus titre with siRNA1 is substantial considering the transfection efficiency and fixed level of input siRNA, siRNA2 emerged to be a better choice as target where more than 300-fold reduction was observed and its inhibitory effect extended up to 48 h post-infection against all the serotypes. Interestingly, in the present study type A virus (IND 17/77) had a single mismatch at position 2 in the siRNA2 target region but it did not abrogate the inhibitory effect.

  16. Cloning and characterisation of mtDBP, a DNA-binding protein which binds two distinct regions of sea urchin mitochondrial DNA.

    PubMed

    Loguercio Polosa, P; Roberti, M; Musicco, C; Gadaleta, M N; Quagliariello, E; Cantatore, P

    1999-04-15

    The cDNA for the sea urchin mitochondrial D-loop-binding protein (mtDBP), a 40 kDa protein which binds two homologous regions of mitochondrial DNA (the D-loop region and the boundary between the oppositely transcribed ND5 and ND6 genes), has been cloned. Four different 3'-untranslated regions have been detected that are related to each other in pairs and do not contain the canonical polyadenylation signal. The in vitro synthesised mature protein (348 amino acids), deprived of the putative signal sequence, binds specifically to its DNA target sequence and produces a DNase I footprint identical to that given by the natural protein. mtDBP contains two leucine zippers, one of which is bipartite, and two small N- and C-terminal basic domains. A deletion mutation analysis of the recombinant protein has shown that the N-terminal region and the two leucine zippers are necessary for the binding. Furthermore, evidence was provided that mtDBP binds DNA as a monomer. This rules out a dimerization role for the leucine zippers and rather suggests that intramolecular interactions between leucine zippers take place. A database search has revealed as the most significative homology a match with the human mitochondrial transcription termination factor (mTERF), a protein that also binds DNA as a monomer and contains three leucine zippers forming intramolecular interactions. These similarities, and the observation that mtDBP-binding sites contain the 3'-ends of mtRNAs coded by opposite strands and the 3'-end of the D-loop structure, point to a dual function of the protein in modulating sea urchin mitochondrial DNA transcription and replication.

  17. A miRNA-binding site single nucleotide polymorphism in the 3'-UTR region of the NOD2 gene is associated with colorectal cancer.

    PubMed

    Ahangari, Fatemeh; Salehi, Rasoul; Salehi, Mansour; Khanahmad, Hosein

    2014-09-01

    Colorectal cancer (CRC) is one of the common malignancies worldwide. Single nucleotide polymorphisms in miRNA-binding site on gene transcripts are reported to play important role in increased risk of CRC in different population. We performed a case-control study using 88 CRC patients and 88 non-cancer counterparts to evaluate the association between NOD2 rs3135500 polymorphism located at 3' untranslated region of the gene and risk of sporadic CRC. Genotyping of rs3135500 polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. We found a significant association of AA genotype with risk of CRC (adjusted OR 3.100, CI 1.621-5.930, p < 0.001). Also, significant difference in physical activity (p = 0.001) between case and control groups was found. We also found that individuals in control group were more aspirin or NSAID user compared to sporadic CRC cases (p = 0.002). In the case group, individuals with GG genotype consumed more aspirin or NSAID compared with AA+AG genotypes (33.3 vs. 9.6 %, adjusted OR 4.71, CI 1.25-17.76, p = 0.02). However, in the control group, individuals with AA+AG genotypes used more aspirin or NSAID compared with GG genotypes (47.2 vs. 11.4 %, adjusted OR 14 %, CI 0.05-0.47, p < 0.001).

  18. Landslides of Palestinian Region

    NASA Astrophysics Data System (ADS)

    Alwahsh, H.

    2013-12-01

    Natural disasters are extreme sudden events caused by environmental and natural actors that take away the lives of many thousands of people each year and damage large amount of properties. They strike anywhere on earth, often without any warning. A risk maps of natural disaster are very useful to identify the places that might be adversely affected in the event of natural disaster. The earthquakes are one of natural disaster that have the greatest hazards and will cause loss of life and properties due to damaging the structures of building, dams, bridges. In addition, it will affect local geology and soil conditions. The site effects play an important role in earthquake risk because of its amplification or damping simulation. Another parameter in developing risk map is landslide, which is also one of the most important topics in site effect hazards. Palestine region has been suffering landslide hazards because of the topographical and geological conditions of this region. Most Palestine consists of mountainous area, which has great steep slopes and the type of soil is mainly grayish to yellowish silty clay (Marl Soil). Due to the above mentioned factors many landslides have been occurred from Negev south to the northern borders of Palestine. An example of huge and destruction landslide in a Palestine authority is the landslide in the White Mountain area in the city of Nablus, which occurred in 1997. The geotechnical and geophysical investigation as well as slope stability analysis should be considered in making landslide maps that are necessary to develop risk levels of the natural disaster. Landslides occurred in slopes that are created naturally or by human beings. Failure of soil mass occurs, and hence landslide of soil mass happen due to sliding of soil mass along a plane or curved surface. In general, the slopes become unstable when the shear stresses (driving force) generated in the soil mass exceed the available shearing resistance on the rupture surface

  19. Sudurnes Regional Heating Corp.

    SciTech Connect

    Lienau, P.J.

    1996-11-01

    The Svartsengi geothermal area is close to the town of Grindavik on the Rekjanes peninsula and is part of an active fissure swarm, lined with crater-rows and open fissures and faults. The high-temperature area has an area of 2 sq. km and shows only limited signs of geothermal activity at the surface. The reservoir, however, contains lots of energy and at least 8 wells supply the Svartsengi Power Plant with steam. The steam is not useable for domestic heating purposes so that heat exchangers are used to heat cold groundwater with the steam. Some steam is also used for producing 16.4 MW{sub e} of electrical power. The article shows the distribution system piping hot water to nine towns and the Keflavik International Airport. The effluent brine from the Svartsengi Plant is disposed of into a surface pond, called the Blue Lagoon, popular to tourists and people suffering from psoriasis and other forms of eczema seeking therapeutic effects from the silica rich brine. This combined power plant and regional district heating system (cogeneration) is an interesting and unique design for the application of geothermal energy.

  20. Active region flows

    NASA Technical Reports Server (NTRS)

    Foukal, Peter

    1987-01-01

    A wide range of observations has shown that active region phenomena in the photospheric, chromospheric and coronal temperature regimes are dynamical in nature. At the photosphere, recent observations of full line profiles place an upper limit of about + or - 20/msec on any downflows at supergranule cell edges. Observations of the full Stokes 5 profiles in the network show no evidence for downflows in magnetic flux tubes. In the area of chromospheric dynamics, several models were put forward recently to reproduce the observed behavior of spicules. However, it is pointed out that these adiabatic models do not include the powerful radiative dissipation which tend to damp out the large amplitude disturbances that produce the spicular acceleration in the models. In the corona, loop flows along field lines clearly transport mass and energy at rates important for the dynamics of these structures. However, advances in understanding the heating and mass balance of the loop structures seem to require new kinds of observations. Some results are presented using a remote sensing diagnostic of the intensity and orientation of macroscopic plasma electric fields predicted by models of reconnective heating and also wave heating.

  1. Regional Kendall test for trend

    USGS Publications Warehouse

    Helsel, D.R.; Frans, L.M.

    2006-01-01

    Trends in environmental variables are often investigated within a study region at more than one site. At each site, a trend analysis determines whether a trend has occurred. Yet often also of interest is whether a consistent trend is evident throughout the entire region. This paper adapts the Seasonal Kendall trend test to determine whether a consistent regional trend occurs in environmental variables.

  2. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  3. Molecular analysis of functional and nonfunctional genes for human ferrochelatase: Isolation and characterization of a FECH pseudogene and its sublocation on chromosome 3

    SciTech Connect

    Whitcombe, D.M.; Albertson, D.G. ); Cox, T.M. )

    1994-04-01

    A pseudogene related to the functional gene (FECH) for the heme biosynthetic enzyme ferrochelatase (ferroheme-protolyase; EC 4.99.1.1.) was isolated from a human genomic library using a ferrochelatase cDNA hybridization probe. The pseudogene shows >80% overall nucleotide sequence identity to the functional gene (including the 3[prime] untranslated region and polyadenylation signals) but contains no intronic sequences in the region corresponding to the open reading frame of expressed ferrochelatase. Furthermore, the pseudogene sequence contains small deletions and insertions creating frameshifts and numerous termination codons, indicating that it does not encode a functional polypeptide. Northern blot analysis using pseudogene-specific probes failed to demonstrate transcripts in samples of human erythroid cell RNA in which ferrochelatase mRNA was readily detected. Southern blot experiments using restriction endonuclease-digested human genomic DNA probed either with ferrochelatase-specific cDNA fragments or pseudogene-specific genomic sequences confirmed the presence of distinct loci for the expressed and nonfunctional genes, respectively. Localization of the human ferrochelatase pseudogene to 3p22-p23 was determined by fluorescent metaphase chromosomal hybridization in situ using three genomic clones in [lambda]EMBL3 spanning a contiguous region of [approximately] 30 kb. This newly identified locus, distinct from the expressed FECH gene, on 18q22, is characteristic of a processed human pseudogene. The existence of the ferrochelatase pseudogene has practical implications for the molecular analysis of mutations responsible for erythropoietic protoporphyria in man. 24 refs., 3 figs.

  4. Regions. [Africa, Middle East].

    PubMed

    1985-03-01

    This discussion of population focuses on the regions of Africa and the Middle East. In South Africa more white women are working but fewer black women work. The overall result is that the percentage of women who work is declining. Marita de Beer, research liaison executive at the South African Advertising Research Foundation, reports that the female population grew by 31% in the past 10 years while the number of working women has grown by only 11%. Among blacks the female population rose by 36%, but the number of workers among them declined by about 1%. Married women are among the fastest growing groups of working women in South Africa. The most recent estimate of the population of Nigeria is 92 million. According to Professor Vremudia Diejomaoh, Nigeria's population will probably reach 155 million by 2000 with 33% living in urban areas. In Saudi Arabia the Pan Arab Research Center recently completed a census of retail outlets in 3 metropolitan areas: Jeddah, Riyadh, and Dammam. The types of outlets surveyed include large supermarkets, small supermarkets, groceries with and without deep freeze, tobacco shops, meat shop/delis, small cafeterias, large restaurants/hotels, cosmetics shops or perfumeries, camera stores, toy shops, pharmacies, watch and gift shop, newsstands, department store, and appliance outlets. Using the Census of Retail Outlets as a base, Pan Arab Research Center also has a new distribution audit system that will cover 500 outlets. By plotting Arab countries according to their population policies and their current growth rates, it is possible to project where the middle class will grow fastest in the Arab world. The countries that have declining growth rates and strong population programs designed to encourage lower fertility rates among women are Egypt, Tunisia, Morocco, Algeria, and Lebanon. The countries most likely to have a better per capita distribution of resources within this decade are those where governments encourage reductions in

  5. Regions. [Africa, Middle East].

    PubMed

    1985-03-01

    This discussion of population focuses on the regions of Africa and the Middle East. In South Africa more white women are working but fewer black women work. The overall result is that the percentage of women who work is declining. Marita de Beer, research liaison executive at the South African Advertising Research Foundation, reports that the female population grew by 31% in the past 10 years while the number of working women has grown by only 11%. Among blacks the female population rose by 36%, but the number of workers among them declined by about 1%. Married women are among the fastest growing groups of working women in South Africa. The most recent estimate of the population of Nigeria is 92 million. According to Professor Vremudia Diejomaoh, Nigeria's population will probably reach 155 million by 2000 with 33% living in urban areas. In Saudi Arabia the Pan Arab Research Center recently completed a census of retail outlets in 3 metropolitan areas: Jeddah, Riyadh, and Dammam. The types of outlets surveyed include large supermarkets, small supermarkets, groceries with and without deep freeze, tobacco shops, meat shop/delis, small cafeterias, large restaurants/hotels, cosmetics shops or perfumeries, camera stores, toy shops, pharmacies, watch and gift shop, newsstands, department store, and appliance outlets. Using the Census of Retail Outlets as a base, Pan Arab Research Center also has a new distribution audit system that will cover 500 outlets. By plotting Arab countries according to their population policies and their current growth rates, it is possible to project where the middle class will grow fastest in the Arab world. The countries that have declining growth rates and strong population programs designed to encourage lower fertility rates among women are Egypt, Tunisia, Morocco, Algeria, and Lebanon. The countries most likely to have a better per capita distribution of resources within this decade are those where governments encourage reductions in

  6. Europa Wedge Region

    NASA Technical Reports Server (NTRS)

    1998-01-01

    This image shows an area of crustal separation on Jupiter's moon, Europa. Lower resolution pictures taken earlier in the tour of NASA's Galileo spacecraft revealed that dark wedge-shaped bands in this region are areas where the icy crust has completely pulled apart. Dark material has filled up from below and filled the void created by this separation.

    In the lower left corner of this image, taken by Galileo's onboard camera on December 16, 1997, a portion of one dark wedge area is visible, revealing a linear texture along the trend of the wedge. The lines of the texture change orientation slightly and reflect the fact that we are looking at a bend in the wedge. The older, bright background, visible on the right half of the image, is criss-crossed with ridges. A large, bright ridge runs east-west through the upper part of the image, cutting across both the older background plains and the wedge. This ridge is rough in texture, with numerous small terraces and troughs containing dark material.

    North is to the top of the picture and the sun illuminates the surface from the northwest. This image, centered at approximately 16.5 degrees south latitude and 196.5 degrees west longitude, covers an area approximately 10 kilometers square (about 6.5 miles square). The resolution of this image is about 26 meters per picture element. This image was taken by the solid state imaging system from a distance of 1250 kilometers (750 miles).

    The Jet Propulsion Laboratory, Pasadena, CA manages the Galileo mission for NASA's Office of Space Science, Washington, DC. JPL is an operating division of California Institute of Technology (Caltech).

    This image and other images and data received from Galileo are posted on the World Wide Web, on the Galileo mission home page at URL http://www.jpl.nasa.gov/ galileo.

  7. Relative effects of mutability and selection on single nucleotide polymorphisms in transcribed regions of the human genome

    PubMed Central

    Gorlov, Ivan P; Gorlova, Olga Y; Amos, Christopher I

    2008-01-01

    Motivation Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in humans. However, the factors that affect SNP density are poorly understood. The goal of this study was to estimate the relative effects of mutability and selection on SNP density in transcribed regions of human genes. It is important for prediction of the regions that harbor functional polymorphisms. Results We used frequency-validated SNPs resulting from single-nucleotide substitutions. SNPs were subdivided into five functional categories: (i) 5' untranslated region (UTR) SNPs, (ii) 3' UTR SNPs, (iii) synonymous SNPs, (iv) SNPs producing conservative missense mutations, and (v) SNPs producing radical missense mutations. Each of these categories was further subdivided into nine mutational categories on the basis of the single-nucleotide substitution type. Thus, 45 functional/mutational categories were analyzed. The relative mutation rate in each mutational category was estimated on the basis of published data. The proportion of segregating sites (PSSs) for each functional/mutational category was estimated by dividing the observed number of SNPs by the number of potential sites in the genome for a given functional/mutational category. By analyzing each functional group separately, we found significant positive correlations between PSSs and relative mutation rates (Spearman's correlation coefficient, at least r = 0.96, df = 9, P < 0.001). We adjusted the PSSs for the mutation rate and found that the functional category had a significant effect on SNP density (F = 5.9, df = 4, P = 0.001), suggesting that selection affects SNP density in transcribed regions of the genome. We used analyses of variance and covariance to estimate the relative effects of selection (functional category) and mutability (relative mutation rate) on the PSSs and found that approximately 87% of variation in PSS was due to variation in the mutation rate and approximately 13% was due to selection

  8. Emission measure distribution for diffuse regions in solar active regions

    SciTech Connect

    Subramanian, Srividya; Tripathi, Durgesh; Klimchuk, James A.; Mason, Helen E.

    2014-11-01

    Our knowledge of the diffuse emission that encompasses active regions is very limited. In this paper we investigate two off-limb active regions, namely, AR 10939 and AR 10961, to probe the underlying heating mechanisms. For this purpose, we have used spectral observations from Hinode/EIS and employed the emission measure (EM) technique to obtain the thermal structure of these diffuse regions. Our results show that the characteristic EM distributions of the diffuse emission regions peak at log T = 6.25 and the coolward slopes are in the range 1.4-3.3. This suggests that both low- as well as high-frequency nanoflare heating events are at work. Our results provide additional constraints on the properties of these diffuse emission regions and their contribution to the background/foreground when active region cores are observed on-disk.

  9. Callisto's Equatorial Region

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This mosaic covers part of the equatorial region of Jupiter's moon, Callisto. The mosaic combines six separate image frames obtained by the solid state imaging (CCD) system on NASA's Galileo spacecraft during its ninth orbit around Jupiter. North is to the top of the picture. The mosaic shows several new features and characteristics of the surface revealed by Galileo. These include deposits that may represent landslides in the southern and southwestern floors of many craters. Two such deposits are seen in a 12 kilometer (7.3 mile) crater in the west-central part of the image, and in a 23 kilometer (14 mile) crater just north of the center of the image. Also notable are several sinuous valleys emanating from the southern rims of 10 to 15 kilometer (6.2 to 9.3 mile) irregular craters in the west-central part of the image. The pervasive local smoothing of Callisto's surface is well represented in the plains between the craters in the southeastern part of the image. Possible oblique impacts are suggested by the elongated craters in the northeastern and southeastern parts of the image.

    The mosaic, centered at 7.4 degrees south latitude and 6.6 degrees west longitude, covers an area of approximately 315 by 215 kilometers (192 by 131 miles). The sun illuminates the scene from the west (left). The smallest features that can be seen are about 300 meters (993 feet) across. The images were obtained on June 25, 1997, when the spacecraft was at a range of 15,200 kilometers (8,207 miles) from Callisto.

    The Jet Propulsion Laboratory, Pasadena, CA manages the Galileo mission for NASA's Office of Space Science, Washington, DC.

    This image and other images and data received from Galileo are posted on the World Wide Web, on the Galileo mission home page at URL http://galileo.jpl.nasa.gov. Background information and educational context for the images can be found at URL http://www.jpl.nasa.gov/galileo/sepo

  10. Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

    PubMed

    Fellah, J S; Wiles, M V; Charlemagne, J; Schwager, J

    1992-10-01

    cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences. PMID:1382992

  11. Sex-specific associations of variants in regulatory regions of NADPH oxidase-2 (CYBB) and glutathione peroxidase 4 (GPX4) genes with kidney disease in type 1 diabetes.

    PubMed

    Monteiro, M B; Patente, T A; Mohammedi, K; Queiroz, M S; Azevedo, M J; Canani, L H; Parisi, M C; Marre, M; Velho, G; Corrêa-Giannella, M L

    2013-10-01

    Oxidative stress is involved in the pathophysiology of diabetic nephropathy. The superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2, encoded by the CYBB gene) and the antioxidant enzyme glutathione peroxidase 4 (GPX4) play opposing roles in the balance of cellular redox status. In the present study, we investigated associations of single nucleotide polymorphisms (SNPs) in the regulatory regions of CYBB and GPX4 with kidney disease in patients with type 1 diabetes. Two functional SNPs, rs6610650 (CYBB promoter region, chromosome X) and rs713041 (GPX4 3'untranslated region, chromosome 19), were genotyped in 451 patients with type 1 diabetes from a Brazilian cohort (diabetic nephropathy: 44.6%) and in 945 French/Belgian patients with type 1 diabetes from Genesis and GENEDIAB cohorts (diabetic nephropathy: 62.3%). The minor A-allele of CYBB rs6610650 was associated with lower estimated glomerular filtration rate (eGFR) in Brazilian women, and with the prevalence of established/advanced nephropathy in French/Belgian women (odds ratio 1.75, 95% CI 1.11-2.78, p = 0.016). The minor T-allele of GPX4 rs713041 was inversely associated with the prevalence of established/advanced nephropathy in Brazilian men (odds ratio 0.30, 95% CI 0.13-0.68, p = 0.004), and associated with higher eGFR in French/Belgian men. In conclusion, these heterogeneous results suggest that neither CYBB nor GPX4 are major genetic determinants of diabetic nephropathy, but nevertheless, they could modulate in a gender-specific manner the risk for renal disease in patients with type 1 diabetes. PMID:23919599

  12. Sex-specific associations of variants in regulatory regions of NADPH oxidase-2 (CYBB) and glutathione peroxidase 4 (GPX4) genes with kidney disease in type 1 diabetes.

    PubMed

    Monteiro, M B; Patente, T A; Mohammedi, K; Queiroz, M S; Azevedo, M J; Canani, L H; Parisi, M C; Marre, M; Velho, G; Corrêa-Giannella, M L

    2013-10-01

    Oxidative stress is involved in the pathophysiology of diabetic nephropathy. The superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2, encoded by the CYBB gene) and the antioxidant enzyme glutathione peroxidase 4 (GPX4) play opposing roles in the balance of cellular redox status. In the present study, we investigated associations of single nucleotide polymorphisms (SNPs) in the regulatory regions of CYBB and GPX4 with kidney disease in patients with type 1 diabetes. Two functional SNPs, rs6610650 (CYBB promoter region, chromosome X) and rs713041 (GPX4 3'untranslated region, chromosome 19), were genotyped in 451 patients with type 1 diabetes from a Brazilian cohort (diabetic nephropathy: 44.6%) and in 945 French/Belgian patients with type 1 diabetes from Genesis and GENEDIAB cohorts (diabetic nephropathy: 62.3%). The minor A-allele of CYBB rs6610650 was associated with lower estimated glomerular filtration rate (eGFR) in Brazilian women, and with the prevalence of established/advanced nephropathy in French/Belgian women (odds ratio 1.75, 95% CI 1.11-2.78, p = 0.016). The minor T-allele of GPX4 rs713041 was inversely associated with the prevalence of established/advanced nephropathy in Brazilian men (odds ratio 0.30, 95% CI 0.13-0.68, p = 0.004), and associated with higher eGFR in French/Belgian men. In conclusion, these heterogeneous results suggest that neither CYBB nor GPX4 are major genetic determinants of diabetic nephropathy, but nevertheless, they could modulate in a gender-specific manner the risk for renal disease in patients with type 1 diabetes.

  13. Regional governance: strategies and disputes in health region management

    PubMed Central

    dos Santos, Adriano Maia; Giovanella, Ligia

    2014-01-01

    OBJECTIVE To analyze the regional governance of the health systemin relation to management strategies and disputes. METHODOLOGICAL PROCEDURES A qualitative study with health managers from 19 municipalities in the health region of Bahia, Northeastern Brazil. Data were drawn from 17 semi-structured interviews of state, regional, and municipal health policymakers and managers; a focus group; observations of the regional interagency committee; and documents in 2012. The political-institutional and the organizational components were analyzed in the light of dialectical hermeneutics. RESULTS The regional interagency committee is the chief regional governance strategy/component and functions as a strategic tool for strengthening governance. It brings together a diversity of members responsible for decision making in the healthcare territories, who need to negotiate the allocation of funding and the distribution of facilities for common use in the region. The high turnover of health secretaries, their lack of autonomy from the local executive decisions, inadequate technical training to exercise their function, and the influence of party politics on decision making stand as obstacles to the regional interagency committee’s permeability to social demands. Funding is insufficient to enable the fulfillment of the officially integrated agreed-upon program or to boost public supply by the system, requiring that public managers procure services from the private market at values higher than the national health service price schedule (Brazilian Unified Health System Table). The study determined that “facilitators” under contract to health departments accelerated access to specialized (diagnostic, therapeutic and/or surgical) services in other municipalities by direct payment to physicians for procedure costs already covered by the Brazilian Unified Health System. CONCLUSIONS The characteristics identified a regionalized system with a conflictive pattern of governance and

  14. Document Delivery Policy. Region 2 [Regional Medical Library Network].

    ERIC Educational Resources Information Center

    Southeastern/Atlantic Regional Medical Library Services, Baltimore, MD.

    Standardized policies and procedures for interlibrary loan and resource sharing in the Southeastern/Atlantic Region of the Regional Medical Library (RML) Network are presented in this policy statement. RML network institutions, which are divided into categories based on their ability and willingness to assume responsibility for interlibrary…

  15. 17 CFR 140.2 - Regional office-regional coordinators.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... direction of a Regional Coordinator who, as a collateral duty, oversees the administration of the office and... parties. Each regional office has delegated authority for the enforcement of the Act and administration of... administration of programs of the Commission in the States of Alabama, Connecticut, Delaware, Florida,...

  16. Isolation, expression, and chromosomal localization of the human mitochondrial capsule selenoprotein gene (MCSP)

    SciTech Connect

    Aho, Hanne; Schwemmer, M.; Tessmann, D.; Murphy, D.

    1996-03-01

    The mitochondrial capsule selenoprotein (MCS) (HGMW-approved symbol MCSP) is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. We describe here the isolation of a cDNA, the exon-intron organization, the expression, and the chromosomal localization of the human MCS gene. Nucleotide sequence analysis of the human and mouse MCS cDNAs reveals that the 5{prime}- and 3{prime}-untranslated sequences are more conserved (71%) than the coding sequences (59%). The open reading frame encodes a 116-amino-acid protein and lacks the UGA codons, which have been reported to encode the selenocysteines in the N-terminal of the deduced mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein (39%). The most striking homology lies in the dicysteine motifs. Northern and Southern zooblot analyses reveal that the MCS gene in human, baboon, and bovine is more conserved than its counterparts in mouse and rat. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5{prime}-untranslated region at a position equivalent to that in the mouse and rat genes. Northern blot and in situ hybridization experiments demonstrate that the expression of the human MCS gene is restricted to haploid spermatids. The human gene was assigned to q21 of chromosome 1. 30 refs., 9 figs.

  17. Coamplification in tumors of KRAS2, type 2 inositol 1,4,5 triphosphate receptor gene, and a novel human gene, KRAG

    SciTech Connect

    Heighway, J.; Betticher, D.C.; Altermatt, H.J.

    1996-07-01

    Analysis of a region of DNA, coamplified in tumors with KRAS2, resulted in the identification of the human homologue of the mouse KRAG gene. The gene was widely expressed in range of cell lines, tumors, and normal tissue and demonstrated a high degree of alternate splicing. A human KRAG cDNA sequence, with a structure similar to that encoded by the amplified gene in mouse Y1 adrenal carcinoma cells, was isolated by RT-PCR. The predicted amino acid similarity between the two sequences was 91%, and hydrophobicity plots suggested a structure closely resembling that of transmembrane 4 superfamily members. Identification of a PCR-based restriction fragment length polymorphism allele-specific splicing differences in tumors. Northern analysis of mRNA derived from a range of tissues suggested high level expression in muscle and confirmed alternate splicing. To facilitate the analysis of exon junctions, a YAC clone encoding the genomic sequence was identified. This allowed the localization of KRAG to human chromosome 12p11.2. Isolation of one end of this nonchimeric clone demonstrated a perfect match with a 247-bp sequence within the 3{prime} untranslated region of the type 2 1,4,5-inositol triphosphate receptor gene. Multiplex PCR confirmed the inclusion of both genes. Multiplex PCR confirmed the inclusion of both genes in the KRAS2 amplicon in human malignancy, suggesting that either may contribute to the malignant phenotypes. 35 refs., 6 figs., 1 tab.

  18. Expression of the human amylase genes: Recent origin of a salivary amylase promoter from an actin pseudogene

    SciTech Connect

    Samuelson, L.C.; Gumucio, D.L.; Meisler, M.H. ); Wiebauer, K. )

    1988-09-12

    The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. The authors have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5{prime} regions of the five human amylase genes contain a processed {gamma}-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3{prime} untranslated region of {gamma}-actin. In addition, insertion of an endogenous retrovirus has interrupted the {gamma}-actin pseudogene in four of the five amylase genes.

  19. Localization of the human fibromodulin gene (FMOD) to chromosome 1q32 and completion of the cDNA sequence

    SciTech Connect

    Sztrolovics, R.; Grover, J.; Roughley, P.J.

    1994-10-01

    This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig.

  20. Identical homologs of the Galanthus nivalis agglutinin in Zea mays and Fusarium verticillioides.

    PubMed

    Fouquaert, Elke; Peumans, Willy J; Gheysen, Godelieve; Van Damme, Els J M

    2011-01-01

    The structural domain corresponding to the Galanthus nivalis agglutinin (GNA) is a mannose-binding motif that was originally discovered in plants but according to recent data also occurs in other eukaryotes and prokaryotes. Transcriptome analyses revealed that Fusarium verticillioides expresses a protein (FvGLLc1) identical to a recently identified cytoplasmic/nuclear GNA-like lectin from maize (ZmGLLc). The FvGLLc1 and ZmGLLc gene sequences are nearly identical in the coding region as well as in the intron and the 5 and 3 prime untranslated regions. However, whereas the Fusarium genome contains only a single gene with an intron, both an intronless and an intron containing lectin gene can be amplified from maize DNA. Southern blot analysis confirmed the presence of this cytoplasmic GNA-like gene in the maize and rice genome. A comparative analysis of the products amplified by different PCRs using genomic DNA from Fusarium species and maize DNA samples from sterile as well as contaminated plant material strongly indicated that the GNA-like sequence found in maize grown under sterile conditions is not derived from a contaminating Fusarium species. Furthermore, using a PCR-based approach it could be demonstrated that this particular type of lectin occurs also in other plants from distant taxa and is markedly conserved.

  1. Genomic organization of the human gene (CA5) and pseudogene for mitochondrial carbonic anhydrase V and their localization to chromosomes 16q and 16p

    SciTech Connect

    Nagao, Yoshiro; Sly, W.S.; Batanian, J.R.

    1995-08-10

    Carbonic anhydrase V (CA V) is expressed in mitochondrial matrix in liver and several other tissues. It is of interest for its putative roles in providing bicarbonate to carbamoyl phosphate synthetase for ureagenesis and to pyruvate carboxylase for gluconeogenesis and its possible importance in explaining certain inherited metabolic disorders with hyperammonemia and hypoglycemia. Following the recent characterization of the cDNA for human CA V, we report the isolation of the human gene from two {lambda} genomic libraries and its characterization. The CA V gene (CA5) is approximately 50 kb long and contains 7 exons and 6 introns. The exon-intron boundaries are found in positions identical to those determined for the previously described CA II, CA III, and CA VII genes. Like the CA VII gene, CA5 does not contain typical TATA and CAAT promoter elements in the 5{prime} flanking region but does contain a TTTAA sequence 147 nucleotides upstream of the initiation codon. CA5 also contains a 12-bp GT-rich segment beginning 13 bp downstream of the polyadenylation signal in the 3{prime} untranslated region of exon 7. FISH analysis allowed CA5 to be assigned to chromosome 16q24.3. An unprocessed pseudogene containing sequence homologous to exons 3-7 and introns 3-6 was also isolated and was assigned by FISH analysis to chromosome 16p11.2-p12. 22 refs., 4 figs., 1 tab.

  2. Genomic organization of the adrenoleukodystrophy gene

    SciTech Connect

    Sarde, C.O.; Mosser, J.; Kretz, C.

    1994-07-01

    Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, is a severe neurodegenerative disease associated with an impairment of very long chain fatty acids {beta}-oxidation. The authors have recently identified by positional cloning the gene responsible for ALD, located in Xq28. It encodes a new member of the {open_quotes}ABC{close_quotes} superfamily of membrane-associated transporters that shows, in particular, significant homology to the 70-kDa peroxisomal membrane protein (PMP70). They report here a detailed characterization of the ALD gene structure. It extends over 21 kb and consists of 10 exons. To facilitate the detection of mutations in ALD patients, they have determined the intronic sequences flanking the exons as well as the sequence of the 3{prime} untranslated region and of the immediate 5{prime} promoter region. Sequences present in distal exons cross-hybridize strongly to additional sequences in the human genome. The ALD gene has been positioned on a pulsed-field map between DXS15 and the L1CAM gene, about 650 kb upstream from the color pigment genes. The frequent occurrence of color vision anomalies observed in patients with adrenomyeloneuropathy (the adult onset form of ALD) thus does not represent a contiguous gene syndrome but a secondary manifestation of ALD. 37 refs., 6 figs.

  3. Polymorphism and genetic mapping of the human oxytocin receptor gene on chromosome 3

    SciTech Connect

    Michelini, S.; Urbanek, M.; Goldman, D.

    1995-06-19

    Centrally administered oxytocin has been reported to facilitate affiliative and social behaviors, in functional harmony with its well-known peripheral effects on uterine contraction and milk ejection. The biological effects of oxytocin could be perturbed by mutations occurring in the sequence of the oxytocin receptor gene, and it would be of interest to establish the position of this gene on the human linkage map. Therefore we identified a polymorphism at the human oxytocin receptor gene. A portion of the 3{prime} untranslated region containing a 30 bp CA repeat was amplified by polymerase chain reaction (PCR), revealing a polymorphism with two alleles occurring with frequencies of 0.77 and 0.23 in a sample of Caucasian CEPH parents (n = 70). The CA repeat polymorphism we detected was used to map the human oxytocin receptor to chromosome 3p25-3p26, in a region which contains several important genes, including loci for Von Hippel-Lindau disease (VHL) and renal cell carcinoma. 53 refs., 2 figs., 1 tab.

  4. About the REL Pacific Region

    ERIC Educational Resources Information Center

    Regional Educational Laboratory Pacific, 2014

    2014-01-01

    REL Pacific is one of ten Regional Educational Laboratories established and funded by the U.S. Department of Education's Institute of Education Sciences. Their region encompasses approximately 4.9 million square miles and serves seven Pacific island entities, including American Samoa; the Commonwealth of the Northern Mariana Islands; the Federated…

  5. Training Teachers for Regional Colleges.

    ERIC Educational Resources Information Center

    Hla Myint; And Others

    This report presents alternative plans for training teachers for the newly-established Regional Colleges in Burma. The Regional Colleges are three-year postsecondary institutions designed to train middle level technicians to help increase the production of goods and services needed in the Burmese economy. Concentrating on the Hawaii Community…

  6. CLIMATE IMPACTS ON REGIONAL WATER

    EPA Science Inventory

    The New England region (including the 6 New England
    states plus upstate New York) offers a very diverse geography,
    matched by an equally diverse economy and human
    population. Livelihoods throughout the region are based
    on service industries that depend heavily on comm...

  7. What's Happening to Regional Policy?

    ERIC Educational Resources Information Center

    Ravenhall, Mark

    2005-01-01

    Back in November, voters in the North East of England overwhelmingly rejected the move towards an elected regional assembly. The scale of the defeat (three to one) of a Government-backed scheme was a rude awakening for the Office of the Deputy Prime Minister and the range of regional agencies created since 1997. After all, it was felt that the…

  8. Adoption Resource Directory: Region X.

    ERIC Educational Resources Information Center

    1983

    State, regional, and national adoption resources are described in this directory for residents of Region X states (Alaska, Idaho, Oregon, and Washington). Emphasizing the adoption of children with special needs, the directory gives organizational contacts for parents in various stages of the adoption process and mentions resources for social…

  9. REGIONAL VULNERABILITY: A CONCEPTUAL FRAMEWORK

    EPA Science Inventory

    Regional vulnerability assessment, or ReVA, is an approach to place-based ecological risk assessment that is currently under development by the Office of Research and Development of the U.S. Environmental Protection Agency (EPA). The assessment is done at the scale of EPA region...

  10. MISR Regional VBBE Imagery Overview

    Atmospheric Science Data Center

    2016-08-24

    ... View Data  |  Download Data About this Web Site: Visualizations of select MISR Level 3 data for special regional ... Regional products are derived from averaging select Level 1 and Level 2 parameters over daily, monthly, seasonal and annual time periods. ...

  11. Regional Early Childhood Policy Review

    ERIC Educational Resources Information Center

    Evans, Judith

    2008-01-01

    The UNESCO-UNICEF joint regional policy review project was launched in September 2006 with the aim to support the countries of Asia-Pacific region in meeting the first goal of Education For All (EFA) on Early Childhood Care and Education (ECCE) by identifying, documenting and sharing good practices as well as constraints and challenges in early…

  12. Global forcing and regional interactions

    USGS Publications Warehouse

    1992-01-01

    The Climate System Modeling Program (CSMP) sponsored a “Global Forcing and Regional Interaction Workshop” from October 21 to 23, 1991, at Colorado State University's Pingree Park campus, to evaluate the relationship between global climate forcing and the response of the land surface on a regional scale. The general aim of the workshop was to develop specific action plans and preliminary science research strategies for regional-global interactions. Each participant was invited to identify tractable, high pay-off science issues related to global forcing and regional interactions. The workshop, with twenty-six participants about evenly split between atmospheric scientists, hydrologists, and ecologists, was also designed to facilitate a network of collaborators to prepare multidisciplinary research proposals. Discussion also focused on regional climate over the last 200 years and included the influence of atmosphere-land surface processes on natural climate variability. Several major recommendations were made on topics discussed.

  13. Disordered regions in transmembrane proteins.

    PubMed

    Tusnády, Gábor E; Dobson, László; Tompa, Peter

    2015-11-01

    The functions of transmembrane proteins in living cells are widespread; they range from various transport processes to energy production, from cell-cell adhesion to communication. Structurally, they are highly ordered in their membrane-spanning regions, but may contain disordered regions in the cytosolic and extra-cytosolic parts. In this study, we have investigated the disordered regions in transmembrane proteins by a stringent definition of disordered residues on the currently available largest experimental dataset, and show a significant correlation between the spatial distributions of positively charged residues and disordered regions. This finding suggests a new role of disordered regions in transmembrane proteins by providing structural flexibility for stabilizing interactions with negatively charged head groups of the lipid molecules. We also find a preference of structural disorder in the terminal--as opposed to loop--regions in transmembrane proteins, and survey the respective functions involved in recruiting other proteins or mediating allosteric signaling effects. Finally, we critically compare disorder prediction methods on our transmembrane protein set. While there are no major differences between these methods using the usual statistics, such as per residue accuracies, Matthew's correlation coefficients, etc.; substantial differences can be found regarding the spatial distribution of the predicted disordered regions. We conclude that a predictor optimized for transmembrane proteins would be of high value to the field of structural disorder. PMID:26275590

  14. Regional strategies for global leadership.

    PubMed

    Ghemawat, Pankaj

    2005-12-01

    The leaders of such global powerhouses as GE, Wal-Mart, and Toyota seem to have grasped two crucial truths: First, far from becoming submerged by the rising tide of globalization, geographic and other regional distinctions may in fact be increasing in importance. Second, regionally focused strategies, used in conjunction with local and global initiatives, can significantly boost a company's performance. The business and economic data reveal a highly regionalized world. For example, trade within regions, rather than across them, drove the surge of international commerce in the second half of the twentieth century. Regionalization is also apparent in foreign direct investment, companies' international sales, and competition among the world's largest multinationals. Harvard Business School Professor Pankaj Ghemawat says that the most successful companies employ five types of regional strategies in addition to--or even instead of--global ones: home base, portfolio, hub, platform, and mandate. Some companies adopt the strategies in sequence, but the most nimble switch from one to another and combine approaches as their markets and businesses evolve. At Toyota, for example, exports from the home base continue to be substantial even as the company builds up an international manufacturing presence. And as Toyota achieves economies of scale and scope with a strong network of hubs, the company also pursues economies of specialization through interregional mandates. Embracing regional strategies requires flexibility and creativity. A company must decide what constitutes a region, choose the most appropriate strategies, and mesh those strategies with the organization's existing structures. In a world that is neither truly global nor truly local, finding ways of coordinating within and across regions can deliver a powerful competitive advantage. PMID:16334585

  15. Regional strategies for global leadership.

    PubMed

    Ghemawat, Pankaj

    2005-12-01

    The leaders of such global powerhouses as GE, Wal-Mart, and Toyota seem to have grasped two crucial truths: First, far from becoming submerged by the rising tide of globalization, geographic and other regional distinctions may in fact be increasing in importance. Second, regionally focused strategies, used in conjunction with local and global initiatives, can significantly boost a company's performance. The business and economic data reveal a highly regionalized world. For example, trade within regions, rather than across them, drove the surge of international commerce in the second half of the twentieth century. Regionalization is also apparent in foreign direct investment, companies' international sales, and competition among the world's largest multinationals. Harvard Business School Professor Pankaj Ghemawat says that the most successful companies employ five types of regional strategies in addition to--or even instead of--global ones: home base, portfolio, hub, platform, and mandate. Some companies adopt the strategies in sequence, but the most nimble switch from one to another and combine approaches as their markets and businesses evolve. At Toyota, for example, exports from the home base continue to be substantial even as the company builds up an international manufacturing presence. And as Toyota achieves economies of scale and scope with a strong network of hubs, the company also pursues economies of specialization through interregional mandates. Embracing regional strategies requires flexibility and creativity. A company must decide what constitutes a region, choose the most appropriate strategies, and mesh those strategies with the organization's existing structures. In a world that is neither truly global nor truly local, finding ways of coordinating within and across regions can deliver a powerful competitive advantage.

  16. LLNL's Regional Seismic Discrimination Research

    SciTech Connect

    Hanley, W; Mayeda, K; Myers, S; Pasyanos, M; Rodgers, A; Sicherman, A; Walter, W

    1999-07-23

    As part of the Department of Energy's research and development effort to improve the monitoring capability of the planned Comprehensive Nuclear-Test-Ban Treaty international monitoring system, Lawrence Livermore Laboratory (LLNL) is testing and calibrating regional seismic discrimination algorithms in the Middle East, North Africa and Western Former Soviet Union. The calibration process consists of a number of steps: (1) populating the database with independently identified regional events; (2) developing regional boundaries and pre-identifying severe regional phase blockage zones; (3) measuring and calibrating coda based magnitude scales; (4a) measuring regional amplitudes and making magnitude and distance amplitude corrections (MDAC); (4b) applying the DOE modified kriging methodology to MDAC results using the regionalized background model; (5) determining the thresholds of detectability of regional phases as a function of phase type and frequency; (6) evaluating regional phase discriminant performance both singly and in combination; (7) combining steps 1-6 to create a calibrated discrimination surface for each stations; (8) assessing progress and iterating. We have now developed this calibration procedure to the point where it is fairly straightforward to apply earthquake-explosion discrimination in regions with ample empirical data. Several of the steps outlined above are discussed in greater detail in other DOE papers in this volume or in recent publications. Here we emphasize the results of the above process: station correction surfaces and their improvement to discrimination results compared with simpler calibration methods. Some of the outstanding discrimination research issues involve cases in which there is little or no empirical data. For example in many cases there is no regional nuclear explosion data at IMS stations or nearby surrogates. We have taken two approaches to this problem, first finding and using mining explosion data when available, and

  17. Future of multistate regional commissions

    SciTech Connect

    Newman, M.

    1980-04-01

    Multistate regional commissions in the United States have been used since 1965. The largest program has been that of the Appalachian Regional Commission (ARC). Institutional and financial barriers have been the most difficult problems encountered by the ARC and other programs (such as Title V commissions). Despite the imperfect performance of the existing regional commissions, they offer a demonstration that some improvement in governmental performance can be achieved. There is virtual unanimity among the nation's governors that this is the route for Federal state relations to follow. Also, the commission route is viewed privately as the most socially acceptable means to have a beneficial impact on government performance. (SAC)

  18. Risk of childhood asthma is associated with CpG-site polymorphisms, regional DNA methylation and mRNA levels at the GSDMB/ORMDL3 locus

    PubMed Central

    Acevedo, Nathalie; Reinius, Lovisa E.; Greco, Dario; Gref, Anna; Orsmark-Pietras, Christina; Persson, Helena; Pershagen, Göran; Hedlin, Gunilla; Melén, Erik; Scheynius, Annika; Kere, Juha; Söderhäll, Cilla

    2015-01-01

    Single-nucleotide polymorphisms (SNPs) in GSDMB (Gasdermin B) and ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) are strongly associated with childhood asthma, but the molecular alterations contributing to disease remain unknown. We investigated the effects of asthma-associated SNPs on DNA methylation and mRNA levels of GSDMB and ORMDL3. Genetic association between GSDMB/ORMDL3 and physician-diagnosed childhood asthma was confirmed in the Swedish birth-cohort BAMSE. CpG-site SNPs (rs7216389 and rs4065275) showed differences in DNA methylation depending on carrier status of the risk alleles, and were significantly associated with methylation levels in two CpG sites in the 5′ UTR (untranslated region) of ORMDL3. In the Swedish Search study, we found significant differences in DNA methylation between asthmatics and controls in five CpG sites; after adjusting for lymphocyte and neutrophil cell counts, three remained significant: one in IKZF3 [IKAROS family zinc finger 3 (Aiolos); cg16293631] and two in the CpG island (CGI) of ORMDL3 (cg02305874 and cg16638648). Also, cg16293631 and cg02305874 correlated with mRNA levels of ORMDL3. The association between methylation and asthma was independent of the genotype in rs7216389, rs4065275 and rs12603332. Both SNPs and CpG sites showed significant associations with ORMDL3 mRNA levels. SNPs influenced expression independently of methylation, and the residual association between methylation and expression was not mediated by these SNPs. We found a differentially methylated region in the CGI shore of ORMDL3 with six CpG sites less methylated in CD8+ T-cells. In summary, this study supports that there are differences in DNA methylation at this locus between asthmatics and controls; and both SNPs and CpG sites are independently associated with ORMDL3 expression. PMID:25256354

  19. Variant in the 3' region of the IkappaBalpha gene associated with insulin resistance in Hispanic Americans: The IRAS Family Study.

    PubMed

    Miller, Melissa R; Zhang, Weiming; Sibbel, Scott P; Langefeld, Carl D; Bowden, Donald W; Haffner, Steven M; Bergman, Richard N; Norris, Jill M; Fingerlin, Tasha E

    2010-03-01

    The IKKbeta/NF-kappaB pathway is known to play an important role in inflammatory response and has also recently been implicated in the process of insulin resistance. We hypothesized that one or more variants in the IkappaBalpha gene (NFKBIA) or surrounding untranslated regions would be associated with insulin sensitivity (S(I)) in Hispanic-American families. We tested for association between 25 single-nucleotide polymorphisms (SNPs) in and near NFKBIA and S(I) in 981 individuals in 90 Hispanic-American families from the Insulin Resistance Atherosclerosis (IRAS) Family Study. SNP rs1951276 in the 3' flanking region of NFKBIA was associated with S(I) in the San Antonio (SA) sample after adjusting for age, gender, and admixture (uncorrected P = 1.69 x 10(-5); conservative Bonferroni correction P = 3.38 x 10(-4)). Subjects with at least one A allele for NFKBIA rs1951276 had approximately 29% lower S(I) compared to individuals homozygous for the G allele in the SA sample. Although not statistically significant, the effect was in the same direction in the San Luis Valley (SLV) sample alone (P = 0.348) and was significant in the combined SA and SLV samples (P = 5.37 x 10(-4); presence of A allele associated with approximately 20% lower S(I)). In SA, when adjusted for subcutaneous adipose tissue area (SAT, cm(2)), the association was modestly attenuated (P = 1.25 x 10(-3)), but the association remained highly significant after adjustment for visceral adipose tissue area (VAT, cm(2); P = 4.41 x 10(-6)). These results provide corroborating evidence that the NF-kappaB/IKKbeta pathway may mediate obesity-induced insulin resistance in humans.

  20. A novel sodium bicarbonate cotransporter-like gene in an ancient duplicated region: SLC4A9 at 5q31

    PubMed Central

    Lipovich, Leonard; Lynch, Eric D; Lee, Ming K; King, Mary-Claire

    2001-01-01

    Background: Sodium bicarbonate cotransporter (NBC) genes encode proteins that execute coupled Na+ and HCO3- transport across epithelial cell membranes. We report the discovery, characterization, and genomic context of a novel human NBC-like gene, SLC4A9, on chromosome 5q31. Results: SLC4A9 was initially discovered by genomic sequence annotation and further characterized by sequencing of long-insert cDNA library clones. The predicted protein of 990 amino acids has 12 transmembrane domains and high sequence similarity to other NBCs. The 23-exon gene has 14 known mRNA isoforms. In three regions, mRNA sequence variation is generated by the inclusion or exclusion of portions of an exon. Noncoding SLC4A9 cDNAs were recovered multiple times from different libraries. The 3' untranslated region is fragmented into six alternatively spliced exons and contains expressed Alu, LINE and MER repeats. SLC4A9 has two alternative stop codons and six polyadenylation sites. Its expression is largely restricted to the kidney. In silico approaches were used to characterize two additional novel SLC4A genes and to place SLC4A9 within the context of multiple paralogous gene clusters containing members of the epidermal growth factor (EGF), ankyrin (ANK) and fibroblast growth factor (FGF) families. Seven human EGF-SLC4A-ANK-FGF clusters were found. Conclusion: The novel sodium bicarbonate cotransporter-like gene SLC4A9 demonstrates abundant alternative mRNA processing. It belongs to a growing class of functionally diverse genes characterized by inefficient highly variable splicing. The evolutionary history of the EGF-SLC4A-ANK-FGF gene clusters involves multiple rounds of duplication, apparently followed by large insertions and deletions at paralogous loci and genome-wide gene shuffling. PMID:11305939

  1. Characterization of the proximal region of the goat NANOG promoter that is used for monitoring cell reprogramming and early embryo development.

    PubMed

    Guo, Yanjie; Lei, Lei; Ma, Xiaoling; Wang, Huayan

    2014-01-01

    Nanog is a key transcription regulatory molecule that plays an important role in maintaining stem cell pluripotency. However, the molecular features and transcription regulation of the NANOG gene in domestic animals are not well investigated. In this study, the 751-base pairs (bp) fragment of the proximal region of the goat NANOG promoter (GNP), which has a 572-bp promoter sequence retaining multiple transcription binding sites and a 179-bp 5' untranslated region of the goat NANOG gene, was cloned and characterized. The recombinant construct of pGNP-EGFP (enhanced green fluorescent protein) was solely activated in pluripotent cells and could be upregulated by the Oct4/Sox2 complex. The construct was stably transfected into goat fetal fibroblast (GFF) cells that were then used as the recipient cells to generate the induced pluripotent stem (iPS) cells. GNP-directed EGFP expression could be used to monitor the progression of cell reprogramming and the formation of iPS cells. The pGNP-EGFP construct was also delivered into goat oocytes cultured in vitro by microinjection. Interestingly, NANOG expression pattern in early stage goat embryos matured in vitro was asymmetrical. In two-cell embryos, the expression level of NANOG was uneven with one blastomere expressing EGFP and the next blastomere with no expression of EGFP. This was also observed in four-cell embryos. This asymmetrical expression may be due to the heterozygous expression of NANOG because of the quality of embryos and the culture environment. In conclusion, the GNP-EGFP reporter system represents a useful tool to monitor endogenous NANOG activation and for research with goat pluripotent stem cells. PMID:24183713

  2. A regional technology transfer program

    NASA Technical Reports Server (NTRS)

    Chenery, P. J.

    1972-01-01

    The activities of the NC/STRC are reported. The background and organization of the regional dissemination center, and marketing methods are discussed along with the services provided, and available information resources.

  3. MISR Regional UAE2 Products

    Atmospheric Science Data Center

    2013-04-29

    ... those familiar with the MISR Level 1 and 2 products, this maps directly to those products; each Regional product summarizes selected ... Theoretical Basis Documents . Images available on this web site include the following parameters: Image Description ...

  4. Active Region Release Two CMEs

    NASA Video Gallery

    Solar material can be seen blowing off the sun in this video captured by NASA’s Solar Dynamics Observatory (SDO) on the night of Feb. 5, 2013. This active region on the sun sent out two coronal ...

  5. Regions.

    ERIC Educational Resources Information Center

    Moulin-Acevedo, Madeleine; And Others

    1993-01-01

    Includes "From School to Jobs: Africa's Dilemma" (Moulin-Acevedo); "Helping Change in Eastern Europe"; "Recognizing the Dignity of Indigenous Peoples"; "An Employment Plan for Pakistan"; and "Around the Continents." (JOW)

  6. Slot Region Radiation Environment Models

    NASA Astrophysics Data System (ADS)

    Sandberg, Ingmar; Daglis, Ioannis; Heynderickx, Daniel; Evans, Hugh; Nieminen, Petteri

    2013-04-01

    Herein we present the main characteristics and first results of the Slot Region Radiation Environment Models (SRREMs) project. The statistical models developed in SRREMs aim to address the variability of trapped electron and proton fluxes in the region between the inner and the outer electron radiation belt. The energetic charged particle fluxes in the slot region are highly dynamic and are known to vary by several orders of magnitude on both short and long timescales. During quiet times, the particle fluxes are much lower than those found at the peak of the inner and outer belts and the region is considered benign. During geospace magnetic storms, though, this region can fill with energetic particles as the peak of the outer belt is pushed Earthwards and the fluxes can increase drastically. There has been a renewed interest in the potential operation of commercial satellites in orbits that are at least partially contained within the Slot Region. Hence, there is a need to improve the current radiation belt models, most of which do not model the extreme variability of the slot region and instead provide long-term averages between the better-known low and medium Earth orbits (LEO and MEO). The statistical models developed in the SRREMs project are based on the analysis of a large volume of available data and on the construction of a virtual database of slot region particle fluxes. The analysis that we have followed retains the long-term temporal, spatial and spectral variations in electron and proton fluxes as well as the short-term enhancement events at altitudes and inclinations relevant for satellites in the slot region. A large number of datasets have been used for the construction, evaluation and inter-calibration of the SRREMs virtual dataset. Special emphasis has been given on the use and analysis of ESA Standard Radiation Environment Monitor (SREM) data from the units on-board PROBA-1, INTEGRAL, and GIOVE-B due to the sufficient spatial and long temporal

  7. The complete exon-intron structure of the 156-kb human gene NFKB1, which encodes the p105 and p50 proteins of transcription factors NF-{kappa}B and I{kappa}B-{gamma}: Implications for NF-{kappa}B-mediated signal transduction

    SciTech Connect

    Heron, E.; Deloukas, P.; van Loon, A.P.G.M.

    1995-12-10

    The NFKB1 gene encodes three proteins of the NF-{kappa}/Rel and I{kappa}B families: p105, p50, and (in mouse) I{kappa}B-{gamma}. We determined the complete genomic structure of human NFKB1. NFKB1 spans 156 kb and has 24 exons with introns varying between 40,000 and 323 bp in length. Although NFKB2, which encodes p100 and p52, also has 24 exons and has a comparable exon-intron structure, it is 20 times shorter than NFKB1. We propose that the long size of NFKB1 is important for transient activation of NF-{kappa}B complexes containing p50. I{kappa}B-{gamma} corresponds to the carboxyl-terminal half of p105. DNA sequence analysis showed that the 3{prime}-end of human intron 11 and the 5{prime}-end of exon 12 of NFKB1 are colinear with the 5{prime}-untranslated region of mouse I{kappa}B-{gamma} cDNA. I{kappa}B-{gamma} is thus likely to be generated by transcription starting within intron 11 and not by alternative splicing of the mouse mRNA encoding p105 and p50. 71 refs., 5 figs., 1 tab.

  8. COL5A1: Genetic mapping and exclusion as candidate gene in families with nail-patella syndrome, tuberous sclerosis 1, hereditary hemorrhagic telangiectasia, and Ehlers-Danlos syndrome type II

    SciTech Connect

    Greenspan, D.S.; Northrup, H.; Au, K.S.

    1995-02-10

    COL5A1, the gene for the {alpha}1 chain of type V collagen, has been considered a candidate gene for certain diseases based on chromosomal location and/or disease phenotype. We have employed 3{prime}-untranslated region RFLPs to exclude COL5A1 as a candidate gene in families with tuberous sclerosis 1, Ehlers-Danlos syndrome type H, and nail-patella syndrome. In addition, we describe a polymorphic simple sequence repeat (SSR) within a COL5A1 intron. This SSR is used to exclude COL5A1 as a candidate gene in hereditary hemorrhagic telangiectasia (Osler-Rendu-Weber disease) and to add COL5A1 to the existing map of {open_quotes}index{close_quotes} markers of chromosome 9 by evaluation of the COL5A1 locus on the CEPH 40-family reference pedigree set. This genetic mapping places COL5A1 between markers D9S66 and D9S67. 14 refs., 1 fig., 2 tabs.

  9. Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor

    SciTech Connect

    Samal, B.; Sun, Yinghao; Stearns, G.

    1994-02-01

    A novel gene coding for the pre-B-cell colony-enhancing factor (PBEF) has been isolated from a human peripheral blood lymphocyte cDNA library. The expression of this gene is induced by pokeweed mitogen and superinduced by cycloheximide. It is also induced in the T-lymphoblastoid cell line HUT 78 after phorbol ester (phorbol myristate acetate) treatment. The predominant mRNA for PBEF is approximately 2.4 kb long and codes for a 52-kDa secreted protein. The 3{prime} untranslated region of the mRNA has multiple TATT motifs, usually found in cytokine and oncogene messages. The PBEF gene is mainly transcribed in human bone marrow, liver tissue, and muscle. We have expressed PBEF in COS 7 and PA317 cells and have tested the biological activities of the conditioned medium as well as the antibody-purified protein in different in vitro assays. PBEF itself had no activity but synergized the pre-B-cell colony formation activity of stem cell factor and interleukin 7. In the presence of PBEF, the number of pre-B-cell colonies was increased by at least 70% above the amount stimulated by stem cell factor plus interleukin 7. No effect of PBEF was found with cells of myeloid or erythroid lineages. These data define PBEF as a novel cytokine which acts on early B-lineage precursor cells. 33 refs., 8 figs.

  10. Human GluR6 kainate receptor (GRIK2): Molecular cloning, expression, polymorphism, and chromosomal assignment

    SciTech Connect

    Paschen, W.; Blackstone, C.D.; Huganir, R.L. ); Ross, C.A. Max-Planck-Institute for Neurological Research, Koeln )

    1994-04-01

    Glutamate receptors mediate the majority of excitatory neurotransmission in the brain, and molecular cloning studies have revealed several distinct families. Because neuropathological states and possibly human disorders may involve kainate-preferring glutamate receptors, the authors have isolated a cDNA clone for the human GluR6 kainate-preferring receptor. This clone shows a very high sequence similarity with that of the rat, except for a part of the 3[prime] untranslated region in which there is a TAA triplet repeat. When th