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Sample records for gene spacer region

  1. The ribosomal gene spacer region in archaebacteria

    NASA Technical Reports Server (NTRS)

    Achenbach-Richter, L.; Woese, C. R.

    1988-01-01

    Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so. The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.

  2. RISSC: a novel database for ribosomal 16S–23S RNA genes spacer regions

    PubMed Central

    García-Martínez, Jesús; Bescós, Ignacio; Rodríguez-Sala, Jesús Javier; Rodríguez-Valera, Francisco

    2001-01-01

    A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S–23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documen­tation tool for studies on evolution, identification, typing and strain characterization, among others. PMID:11125084

  3. Enterococcus species composition determined by capillary electrophoresis of the groESL gene spacer region DNA.

    PubMed

    Yasuda, M; Paar, J; Doolittle, M; Brochi, J; Pancorbo, O C; Tang, R J; Stoner, R E; Shiaris, M P

    2010-07-01

    Marine recreational beaches are monitored for fecal contamination by Enterococcus spp. (ENT) counts. Although different ENT species in the environment tend to thrive in and originate from distinct hosts, the current monitoring method does not differentiate among species. Time-consuming isolation-based species identification precludes routine analysis of environmental ENT communities. Therefore, an isolation-independent DNA fingerprinting method was developed to characterize environmental ENT communities using DNA length polymorphism of the spacer region between the groES and groEL genes common to most ENT species. Capillary electrophoresis resulted in distinct peak sizes of PCR products that carried polymorphic groESL spacers (300-335 bp in length) among 8 different ENT species (Enterococcus avium, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus mundtii, Enterococcus hirae, Enterococcus faecium, Enterococcus durans, and Enterococcus faecalis). Distortions in true species ratios observed in electropherograms were caused by PCR biases arising in a mixed ENT community DNA template. E. faecalis was overestimated and E. avium and E. faecium were underestimated compared to the original species ratios in the mixed community. The PCR product bias was constant between species, so good approximation of the species ratio in ENT communities is possible. In environmental samples, a high percentage of E. faecalis (96%) together with high total ENT counts were observed in samples collected from a sewer line and from several sites in a storm drain system where sewage leaks were suspected. In contrast, samples with <400 CFU 100 ml-1 ENT were either dominated by E. mundtii or had 4 or more ENT species. The latter ENT community profiles are considered to be signatures of enterococci rarely associated with animals with low or of non-fecal origin.

  4. Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons.

    PubMed

    Tannock, G W; Tilsala-Timisjarvi, A; Rodtong, S; Ng, J; Munro, K; Alatossava, T

    1999-09-01

    Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.

  5. Nuclear ribosomal spacer regions in plant phylogenetics: problems and prospects.

    PubMed

    Poczai, Péter; Hyvönen, Jaakko

    2010-04-01

    The nuclear ribosomal locus coding for the large subunit is represented in tandem arrays in the plant genome. These consecutive gene blocks, consisting of several regions, are widely applied in plant phylogenetics. The regions coding for the subunits of the rRNA have the lowest rate of evolution. Also the spacer regions like the internal transcribed spacers (ITS) and external transcribed spacers (ETS) are widely utilized in phylogenetics. The fact, that these regions are present in many copies in the plant genome is an advantage for laboratory practice but might be problem for phylogenetic analysis. Beside routine usage, the rDNA regions provide the great potential to study complex evolutionary mechanisms, such as reticulate events or array duplications. The understanding of these processes is based on the observation that the multiple copies of rDNA regions are homogenized through concerted evolution. This phenomenon results to paralogous copies, which can be misleading when incorporated in phylogenetic analyses. The fact that non-functional copies or pseudogenes can coexist with ortholougues in a single individual certainly makes also the analysis difficult. This article summarizes the information about the structure and utility of the phylogenetically informative spacer regions of the rDNA, namely internal- and external transcribed spacer regions as well as the intergenic spacer (IGS).

  6. Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNA(Ala) gene segments in Campylobacter lari.

    PubMed

    Hayashi, K; Tazumi, A; Nakanishi, S; Nakajima, T; Matsubara, K; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

    2012-06-01

    Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.

  7. Characterization of internal transcribed spacer (ITS1)-ITS2 region of ribosomal RNA gene from 25 species of Culicoides biting midges (Diptera: Ceratopogonidae) in Japan.

    PubMed

    Matsumoto, Y; Yanase, T; Tsuda, T; Noda, H

    2009-09-01

    We determined nucleotide sequences of the nuclear rDNA internal transcribed spacer (ITS)1-5.8S-ITS2a-2S-ITS2 region in 103 individuals of 25 Culicoides species (Diptera: Ceratopogonidae) from 11 locations in Japan. Ribosomal RNA genes, 5.8S and 2S rDNA, were highly conserved among the species with few variations. The ITS2a region showed length variation among species. Both ITS1 and ITS2 showed highly varied sequences among species. The noticeable indel regions among ITS1 sequences are present in some Culicoides species, separating species into two types having long or short ITS1 region. However, Culicoides cylindratus Kitaoka possesses both types of ITS1 in each individual; these results seem to indicate that the ITS1-long type was the prototype and the short type was produced through deletion in many Culicoides species. One species, belonging to subgenus Avaritia, possessed an Avaritia-specific sequence in ITS1 and phylogenetically formed a monophyletic group. Geographical genotypes in a species were not clear. Species-specific sequence features were observed, enabling molecular identification of Culicoides species.

  8. Comparison of the Sequences of the Internal Transcribed Spacer Regions and PbGP43 Genes of Paracoccidioides brasiliensis from Patients and Armadillos (Dasypus novemcinctus)

    PubMed Central

    Hebeler-Barbosa, Flavia; Morais, Flavia V.; Montenegro, Mario R.; Kuramae, Eiko E.; Montes, Beatriz; McEwen, Juan G.; Bagagli, Eduardo; Puccia, Rosana

    2003-01-01

    Paracoccidioides brasiliensis isolates from 10 nine-banded armadillos (Dasypus novemcinctus) were comparable with 19 clinical isolates by sequence analysis of the PbGP43 gene and ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and by random amplified polymorphic DNA. In this original ITS study, eight isolates differed by one or three sites among five total substitution sites. PMID:14662970

  9. Comparative Sequence Analysis of the tuf and recA Genes and Restriction Fragment Length Polymorphism of the Internal Transcribed Spacer Region Sequences Supply Additional Tools for Discriminating Bifidobacterium lactis from Bifidobacterium animalis

    PubMed Central

    Ventura, Marco; Zink, Ralf

    2003-01-01

    The relationship between Bifidobacterium lactis and Bifidobacterium animalis was examined by comparative analysis of tuf and recA gene sequences and by restriction fragment length polymorphism analysis of their internal 16S-23S transcribed spacer region sequences. The bifidobacterial strains investigated could be divided into two distinct groups within a single species based on the tuf, recA, and 16S-23S spacer region sequence analysis. Therefore, all strains of B. lactis and B. animalis could be unified as the species B. animalis and divided into two subspecies, Bifidobacterium animalis subsp. lactis and Bifidobacterium animalis subsp. animalis. PMID:14660406

  10. Cloning of the 18S rDNA gene, an internal transcribed spacer, and the 5' region of the 28S rDNA gene of Cope's gray treefrog, Hyla chrysoscelis.

    PubMed

    Owens, G; Wiley, J E

    2001-01-01

    The location of rDNA genes on the chromosomes of most species is identical within that species, usually occurring on the same chromosome or chromosomes. This is not the case in Cope's gray treefrog, Hyla chrysoscelis, where the rDNA genes are polymorphic for chromosome location. The occasions leading to this polymorphism have yet to be determined. The first step in understanding the nature of the polymorphism is the characterization of the ribosomal gene array. Here we describe the cloning, sequencing, and confirmation, by fluorescence in situ hybridization, of the 18S rDNA gene, a region which includes the end of the 18S rDNA gene, an internal transcribed spacer, and a portion of the 5' end of the 28S rDNA gene in H. chrysoscelis.

  11. Genetic variations in the beta-tubulin gene and the internal transcribed spacer 2 region of Trichuris species from man and baboons.

    PubMed

    Hansen, Tina V A; Thamsborg, Stig M; Olsen, Annette; Prichard, Roger K; Nejsum, Peter

    2013-08-12

    The whipworm Trichuris trichiura has been estimated to infect 604 - 795 million people worldwide. The current control strategy against trichuriasis using the benzimidazoles (BZs) albendazole (400 mg) or mebendazole (500 mg) as single-dose treatment is not satisfactory. The occurrence of single nucleotide polymorphisms (SNPs) in codons 167, 198 or 200 of the beta-tubulin gene has been reported to convey BZ-resistance in intestinal nematodes of veterinary importance. It was hypothesised that the low susceptibility of T. trichiura to BZ could be due to a natural occurrence of such SNPs. The aim of this study was to investigate whether these SNPs were present in the beta-tubulin gene of Trichuris spp. from humans and baboons. As a secondary objective, the degree of identity between T. trichiura from humans and Trichuris spp. from baboons was evaluated based on the beta-tubulin gene and the internal transcribed spacer 2 region (ITS2). Nucleotide sequences of the beta-tubulin gene were generated by PCR using degenerate primers, specific primers and DNA from worms and eggs of T. trichiura and worms of Trichuris spp. from baboons. The ITS2 region was amplified using adult Trichuris spp. from baboons. PCR products were sequenced and analysed. The beta-tubulin fragments were studied for SNPs in codons 167, 198 or 200 and the ITS2 amplicons were compared with GenBank records of T. trichiura. No SNPs in codons 167, 198 or 200 were identified in any of the analysed Trichuris spp. from humans and baboons. Based on the ITS2 region, the similarity between Trichuris spp. from baboons and GenBank records of T. trichiura was found to be 98 - 99%. Single nucleotide polymorphisms in codon 167, 198 and 200, known to confer BZ-resistance in other nematodes, were absent in the studied material. This study does not provide data that could explain previous reports of poor BZ treatment efficacy in terms of polymorphism in these codons of beta-tubulin. Based on a fragment of the beta

  12. Genetic variations in the beta-tubulin gene and the internal transcribed spacer 2 region of Trichuris species from man and baboons

    PubMed Central

    2013-01-01

    Background The whipworm Trichuris trichiura has been estimated to infect 604 – 795 million people worldwide. The current control strategy against trichuriasis using the benzimidazoles (BZs) albendazole (400 mg) or mebendazole (500 mg) as single-dose treatment is not satisfactory. The occurrence of single nucleotide polymorphisms (SNPs) in codons 167, 198 or 200 of the beta-tubulin gene has been reported to convey BZ-resistance in intestinal nematodes of veterinary importance. It was hypothesised that the low susceptibility of T. trichiura to BZ could be due to a natural occurrence of such SNPs. The aim of this study was to investigate whether these SNPs were present in the beta-tubulin gene of Trichuris spp. from humans and baboons. As a secondary objective, the degree of identity between T. trichiura from humans and Trichuris spp. from baboons was evaluated based on the beta-tubulin gene and the internal transcribed spacer 2 region (ITS2). Methods Nucleotide sequences of the beta-tubulin gene were generated by PCR using degenerate primers, specific primers and DNA from worms and eggs of T. trichiura and worms of Trichuris spp. from baboons. The ITS2 region was amplified using adult Trichuris spp. from baboons. PCR products were sequenced and analysed. The beta-tubulin fragments were studied for SNPs in codons 167, 198 or 200 and the ITS2 amplicons were compared with GenBank records of T. trichiura. Results No SNPs in codons 167, 198 or 200 were identified in any of the analysed Trichuris spp. from humans and baboons. Based on the ITS2 region, the similarity between Trichuris spp. from baboons and GenBank records of T. trichiura was found to be 98 – 99%. Conclusions Single nucleotide polymorphisms in codon 167, 198 and 200, known to confer BZ-resistance in other nematodes, were absent in the studied material. This study does not provide data that could explain previous reports of poor BZ treatment efficacy in terms of polymorphism in these codons of beta

  13. Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region.

    PubMed

    Yang, G; Benson, R; Pelish, T; Brown, E; Winchell, J M; Fields, B

    2010-03-01

    Although the majority of cases of Legionnaires' disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.

  14. The internal transcribed spacer region of belonolaimus (nemata: belonolaimidae).

    PubMed

    Cherry, T; Szalanski, A L; Todd, T C; Powers, T O

    1997-03-01

    Belonolaimus isolates from six U.S. states were compared by restriction endonuclease digestion of amplified first internal transcribed spacer region (ITS1) of the nuclear ribosomal genes. Seven restriction enzymes were selected for evaluation based on restriction sites inferred from the nucleotide sequence of a South Carolina Belonolaimus isolate. Amplified product size from individuals of each isolate was approximately 700 bp. All Midwestern isolates gave distinct restriction digestion patterns. Isolates identified morphologically as Belonolaimus longicaudatus from Florida, South Carolina, and Palm Springs, California, were identical for ITS1 restriction patterns. The correlation between ITS1 restriction patterns and the distribution of B. longicaudatus isolates suggest that the California isolate is a relatively recent introduction into the state.

  15. Comparison of sequencing the D2 region of the large subunit ribosomal RNA gene (MicroSEQ®) versus the internal transcribed spacer (ITS) regions using two public databases for identification of common and uncommon clinically relevant fungal species.

    PubMed

    Arbefeville, S; Harris, A; Ferrieri, P

    2017-09-01

    Fungal infections cause considerable morbidity and mortality in immunocompromised patients. Rapid and accurate identification of fungi is essential to guide accurately targeted antifungal therapy. With the advent of molecular methods, clinical laboratories can use new technologies to supplement traditional phenotypic identification of fungi. The aims of the study were to evaluate the sole commercially available MicroSEQ® D2 LSU rDNA Fungal Identification Kit compared to the in-house developed internal transcribed spacer (ITS) regions assay in identifying moulds, using two well-known online public databases to analyze sequenced data. 85 common and uncommon clinically relevant fungi isolated from clinical specimens were sequenced for the D2 region of the large subunit (LSU) of ribosomal RNA (rRNA) gene with the MicroSEQ® Kit and the ITS regions with the in house developed assay. The generated sequenced data were analyzed with the online GenBank and MycoBank public databases. The D2 region of the LSU rRNA gene identified 89.4% or 92.9% of the 85 isolates to the genus level and the full ITS region (f-ITS) 96.5% or 100%, using GenBank or MycoBank, respectively, when compared to the consensus ID. When comparing species-level designations to the consensus ID, D2 region of the LSU rRNA gene aligned with 44.7% (38/85) or 52.9% (45/85) of these isolates in GenBank or MycoBank, respectively. By comparison, f-ITS possessed greater specificity, followed by ITS1, then ITS2 regions using GenBank or MycoBank. Using GenBank or MycoBank, D2 region of the LSU rRNA gene outperformed phenotypic based ID at the genus level. Comparing rates of ID between D2 region of the LSU rRNA gene and the ITS regions in GenBank or MycoBank at the species level against the consensus ID, f-ITS and ITS2 exceeded performance of the D2 region of the LSU rRNA gene, but ITS1 had similar performance to the D2 region of the LSU rRNA gene using MycoBank. Our results indicated that the MicroSEQ® D2 LSU r

  16. Primers ITS1, ITS2 and ITS4 detect the intraspecies variability in the internal transcribed spacers and 5.8S rRNA gene region in clinical isolates of fungi.

    PubMed

    Korabecná, M; Liska, V; Fajfrlík, K

    2003-01-01

    Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.

  17. Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

    PubMed Central

    Lucchini, R; Sogo, J M

    1992-01-01

    The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer. Images PMID:1406621

  18. Genotyping of Pneumocystis jirovecii isolates from Chinese HIV-infected patients based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes.

    PubMed

    Li, Kai; He, Ai; Cai, Wei Ping; Tang, Xiao Ping; Zheng, Xiao Ying; Li, Zhuo Ya; Zhan, Xi Mei

    2013-01-01

    Genetic diversity of Pneumocystis jirovecii isolates based on internal transcribed spacer (ITS) of the nuclear rRNA locus has previously been reported. The information about ITS genotype and epidemiology of this organism in Chinese human immunodeficiency virus-infected patients has not been available. In this study, 12 bronchoalveolar lavage fluid specimens obtained from HIV-infected patients were analyzed by PCR followed by cloning, sequencing and typing. Three ITS1 genotypes (E, B and 'H') and four ITS2 genotypes (b, g, i and r) as previously reported were identified, the most common of which were E, b and i. Five ITS haplotypes (Eg, Eb, Bi, Er and 'H'r) and 19 new combination types were also identified with the most common types being Eg (four of 12 patients, 10 of 60 clones), Eb (three of 12 patients, 11 of 60 clones) and Bi (three of 12 patients, 10 of 60 clones). Nine patients were found to be co-infected with more than one ITS genotype of P. jirovecii. The prevalence of ITS genotypes in HIV patients from one Chinese hospital did not seem to be significantly different when compared to reports from other countries.

  19. Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

    PubMed

    Cura, Carolina I; Mejía-Jaramillo, Ana M; Duffy, Tomás; Burgos, Juan M; Rodriguero, Marcela; Cardinal, Marta V; Kjos, Sonia; Gurgel-Gonçalves, Rodrigo; Blanchet, Denis; De Pablos, Luis M; Tomasini, Nicolás; da Silva, Alexandre; Russomando, Graciela; Cuba, Cesar A Cuba; Aznar, Christine; Abate, Teresa; Levin, Mariano J; Osuna, Antonio; Gürtler, Ricardo E; Diosque, Patricio; Solari, Aldo; Triana-Chávez, Omar; Schijman, Alejandro G

    2010-12-01

    The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time. Copyright © 2010 Australian Society for Parasitology

  20. Internal transcribed spacer region evolution in Larix and Pseudotsuga (Pinaceae).

    PubMed

    Gernandt, D S; Liston, A

    1999-05-01

    The nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region has been characterized in the sister genera Larix and Pseudotsuga (Pinaceae). Complete sequences were obtained for seven species of Larix from North America and Eurasia and five species of Pseudotsuga from western North America and eastern Asia. ITS region lengths ranged from 1759 to 1770 bp in Larix and from 1564 to 1571 bp in Pseudotsuga. In both genera, ITS1 is three times as long as the 5.8S plus ITS2 and contains subrepeats as observed in other genera of Pinaceae. Secondary structure models predicted that the subrepeats fold into terminal stem and loop domains. ITS polymorphism detected within individuals of Larix and Pseudotsuga suggests a slow rate of concerted evolution among nrDNA loci. Except for the placement of L. sibirica, phylogenetic analyses of the ITS region agreed with previously reported restriction site analyses of Larix and Pseudotsuga. The data were not consistent with phylogenetic hypotheses for Larix based primarily upon ovulate cone characters, failing to support a derivation of the North American L. laricina from a short-bracted Eurasian lineage. The phylogenetic hypothesis did not conflict with a stepping stone model of evolution for Pseudotsuga, but a basal lineage could not be inferred for either genus.

  1. Nucleotide sequence of the internal transcribed spacers and 5.8S region of ribosomal DNA in Pinus pinea L.

    PubMed

    Marrocco, R; Gelati, M T; Maggini, F

    1996-01-01

    The nucleotide sequence of the first internal transcribed spacer (ITS1) belonging to different ribosomal RNA genes from Pinus pinea are reported. The analyzed ITS1 can be distinguished on the basis of their length, being one 2631 bp and the other 271 bp long. Nucleotide comparison of these regions did not show appreciable sequence homology. The larger ITS1 contains five tandem arranged subrepeats with size ranging between 219 bp and 237 bp. The nucleotide sequence of the 5.8S and the ITS2 regions belonging to the larger ribosomal RNA gene are also reported.

  2. 16S-23S rDNA internal transcribed spacer regions in four Proteus species.

    PubMed

    Cao, Boyang; Wang, Min; Liu, Lei; Zhou, Zhemin; Wen, Shaoping; Rozalski, Antoni; Wang, Lei

    2009-04-01

    Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS(Glu) (containing tRNA(Glu (UUC)) gene) and ITS(Ile+Ala) (containing tRNA(Ile (GAU)) and tRNA(Ala (UGC)) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

  3. Characterization of Dermanyssus gallinae (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions.

    PubMed

    Potenza, L; Cafiero, M A; Camarda, A; La Salandra, G; Cucchiarini, L; Dachà, M

    2009-10-01

    In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3'end of 18S and at the 5'start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.

  4. Molecular Systematics of Dictyostelids: 5.8S Ribosomal DNA and Internal Transcribed Spacer Region Analyses▿

    PubMed Central

    Romeralo, María; Escalante, Ricardo; Sastre, Leandro; Lado, Carlos

    2007-01-01

    The variability and adaptability of the amoebae from the class Dictyosteliomycetes greatly complicate their systematics. The nucleotide sequences of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene have been determined for 28 isolates, and their utility to discriminate between different species and genera has been shown. PMID:17056743

  5. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.

    PubMed

    Schoch, Conrad L; Seifert, Keith A; Huhndorf, Sabine; Robert, Vincent; Spouge, John L; Levesque, C André; Chen, Wen

    2012-04-17

    Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

  6. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    PubMed Central

    Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

    2012-01-01

    Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

  7. Unusual features of the sequences of copies of the 16S-23S rRNA internal transcribed spacer regions of Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi arise from horizontal gene transfer events.

    PubMed

    Maslunka, Christopher; Gürtler, Volker; Seviour, Robert

    2015-02-01

    The highly variable nature of the internal transcribed spacer region (ITS) has been claimed to represent an ideal target for designing species-specific probes/primers capable of differentiating between closely related Acinetobacter species. However, several Acinetobacter species contain multiple ITS copies of variable lengths, and these include Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi. This study shows these length variations result from inter-genomic insertion/deletion events (indels) involving horizontal transfer of ITS fragments of other Acinetobacter species and possibly unrelated bacteria, as shown previously by us. In some instances, indel incorporation results in the loss of probe target sites in the recipient cell ITS. In other cases, some indel sequences contain target sites for probes designed from a single ITS sequence to target other Acinetobacter species. Hence, these can generate false positives. The largest of the indels that remove probe sites is 683 bp (labelled bay/i1-0), and it derives from the horizontal transfer of a complete ITS between A. bereziniae BCRC15423(T) and A. baylyi strain ADP1. As a consequence, ITS sequencing or fingerprinting cannot be used to distinguish between the 683 bp ITS in these two strains.

  8. Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species.

    PubMed

    Ibáñez-Escribano, Alexandra; Nogal-Ruiz, Juan José; Arán, Vicente J; Escario, José Antonio; Gómez-Barrio, Alicia; Alderete, J F

    2014-04-01

    The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~83%. There was >40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. Phylogenetic relationships of the genus Phanerochaete inferred from the internal transcribed spacer region

    Treesearch

    Theodorus H. de Koker; Karen K. Nakasone; Jacques Haarhof; Harold H. Burdsall; Bernard J.H. Janse

    2003-01-01

    Phanerochaete is a genus of resupinate homobasidiomycetes that are saprophytic on woody debris and logs. Morphological studies in the past indicated that Phanerochaete is a heterogeneous assemblage of species. In this study the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was used to test the monophyly of the genus Phanerochaete and to infer...

  10. Identification of Medically Important Yeast Species by Sequence Analysis of the Internal Transcribed Spacer Regions

    PubMed Central

    Leaw, Shiang Ning; Chang, Hsien Chang; Sun, Hsiao Fang; Barton, Richard; Bouchara, Jean-Philippe; Chang, Tsung Chain

    2006-01-01

    Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods. PMID:16517841

  11. Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGene IDNS [corrected] software for identification of filamentous fungi in a clinical laboratory.

    PubMed

    Kwiatkowski, Nicole P; Babiker, Wisal M; Merz, William G; Carroll, Karen C; Zhang, Sean X

    2012-07-01

    Filamentous fungal infections have recently increased because of the increasing numbers of immunocompromised hosts. In this study, we evaluated DNA sequencing of the D1/D2 region of the large subunit of the 28S ribosomal RNA gene and the internal transcribed spacer (ITS) region using SmartGene (SG; SmartGene Inc., Raleigh, NC) for the identification of a broad range of commonly encountered filamentous fungi. The SG proofreaders were used to upload, align, and edit fragments, and the resultant sequences were interpreted using the quality-controlled SG database. The results were compared with reference identifications using conventional phenotypic methods or ITS DNA sequences obtained from GenBank if phenotypic identifications were inconclusive. A total of 146 clinical isolates were included in this study, representing 49 different genera. The overall agreements of the D1/D2 and the ITS sequencing methods to reference identification were 97.2% (95% CI, 93.1% to 98.9%) and 97.7% (95% CI, 92.8% to 99.4%), respectively. Of the 146 isolates, 18 (12.3%) did not amplify using the ITS universal primers after repeated attempts and, therefore, could not be sequenced using this target. Correct identification was achieved for 100% (95% CI, 97.4% to 100%) of the isolates when applying both the D1/D2 and ITS targets. In summary, DNA sequencing using SG software provides a rapid, accurate, and reliable tool for the identification of filamentous fungi in a clinical laboratory. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  12. Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica.

    PubMed

    Christensen, H; Møller, P L; Vogensen, F K; Olsen, J E

    2000-01-01

    The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

  13. Phylogenetic analysis of Antrodia species and Antrodia camphorata inferred from internal transcribed spacer region.

    PubMed

    Chiu, Hua-Hsien

    2007-04-01

    The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.

  14. Identification of Aspergillus Species Using Internal Transcribed Spacer Regions 1 and 2

    PubMed Central

    Henry, Travis; Iwen, Peter C.; Hinrichs, Steven H.

    2000-01-01

    Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1–5.8S–ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA. PMID:10747135

  15. BIODEGRADABLE BRANCHED POLYCATIONIC POLYMERS WITH VARYING HYDROPHILIC SPACERS FOR NON-VIRAL GENE DELIVERY

    PubMed Central

    Chew, Sue Anne; Hacker, Michael C.; Saraf, Anita; Raphael, Robert M.; Kasper, F. Kurtis; Mikos, Antonios G.

    2009-01-01

    Biodegradable branched polycationic polymers with varying hydrophilic spacer lengths were synthesized from different triacrylate monomers and the amine monomer 1-(2-aminoethyl)piperazine by Michael addition polymerization. The hydrophilic spacers were varied by the number of ethyleneoxy groups in the triacrylate monomer (E/M) that ranged from 0 to 14. The polymer degradation depended on the spacer length and pH; the amount of ester degraded as determined by 1H-NMR after 14 days was 43.4 ± 2.1% (pH 5.0) and 89.7 ± 1.3% (pH 7.4) for the polymer with 0 E/M compared to 55.7 ± 2.6% (pH 5.0) and 98.5 ± 1.6% (pH 7.4) for the polymer with 14 E/M. Cell viability of rat fibroblasts after exposure to polymer solutions of concentrations up to 1000 μg/mL remained high (above 66.9 ± 12.1% compared to below 7.6 ± 1.1% for polyethylenimine at a concentration of 50 μg/mL or higher) and increased with the spacer length. The polyplexes made with all the synthesized polymers showed higher transfection efficiency (4.5 ± 1.7% to 9.4 ± 2.0%, dependent on the polymer/pDNA weight ratio) with an enhanced green fluorescent protein reporter gene compared to naked pDNA (0.8 ± 0.4%) as quantified by flow cytometry. This study demonstrates that hydrophilic spacers can be incorporated into polycationic polymers to reduce their cytotoxicity and enhance their degradability for non-viral gene delivery. PMID:19678696

  16. Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure

    SciTech Connect

    Shiao, Y.-H. . E-mail: shiao@mail.ncifrcf.gov; Crawford, Erik B.; Anderson, Lucy M.; Patel, Pritesh; Ko, Kinarm

    2005-06-15

    Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

  17. Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA.

    PubMed

    Goodwin, S B; Dunkle, L D; Zismann, V L

    2001-07-01

    ABSTRACT Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.

  18. Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species.

    PubMed

    La Scola, Bernard; Gundi, Vijay A K B; Khamis, Atieh; Raoult, Didier

    2006-03-01

    Acinetobacter species are defined on the basis of several phenotypic characters, results of DNA-DNA homology, and more recently, similarities or dissimilarities in 16S rRNA gene sequences. However, the 16S rRNA gene is not polymorphic enough to clearly distinguish all Acinetobacter species. We used an RNA polymerase beta-subunit gene (rpoB)-based identification scheme for the delineation of species within the genus Acinetobacter, and towards that end, we determined the complete rpoB gene and flanking spacer (rplL-rpoB and rpoB-rpoC) sequences of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. By using complete gene sequences (4,089 bp), we clearly separated all species and grouped them into different clusters. A phylogenetic tree constructed using these sequences was supported by bootstrap values higher than those obtained with 16S rRNA or the gyrB or recA gene. Four pairs of primers enabled us to amplify and sequence two highly polymorphic partial sequences (350 and 450 bp) of the rpoB gene. These and flanking spacers were designed and tested for rapid identification of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. Each of these four variable sequences enabled us to delineate most species. Sequences of at least two polymorphic sequences should be used to distinguish Acinetobacter grimontii, Acinetobacter junii, Acinetobacter baylyi, and genomic species 9 from one another. Finally, 21 clinical isolates of Acinetobacter baumannii were tested for intraspecies relationships and assigned correctly to the same species by comparing the partial sequences of the rpoB gene and its flanking spacers.

  19. Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non-silk-like "spacer regions".

    PubMed

    Colgin, M A; Lewis, R V

    1998-03-01

    Spider minor ampullate silk is a strong non-elastic deformably stretchable silk used in web formation. This silk from Nephila clavipes is composed of two proteins, MiSp 1 and 2, whose transcripts are 9.5 and 7.5 kb, respectively, as determined by Northern blots. Both MiSp proteins are organized into a predominantly repetitive region and a small nonrepetitive carboxy terminal region. These highly repetitive regions are composed mainly of glycine and alanine, but also contain tyrosine, glutamine, and arginine. The sequences are mainly GGX and GA repeats. The repetitive regions are interrupted by nonrepetitive serine-rich spacer regions. Although the sequences of the spacer regions differ from the repetitive regions, sequences of the spacers from different regions of the proteins are nearly identical. The sequence differences between major and minor ampullate silks may explain the differing mechanical properties of the fibers.

  20. Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

    PubMed

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

    2015-10-22

    Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

  1. Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology

    PubMed Central

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K. P.; Woo, Patrick C. Y.

    2015-01-01

    Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10–49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n = 2), Pichia (Candida) norvegensis (n = 2), Candida tropicalis (n = 1) and Saccharomyces cerevisiae (n = 1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study. PMID:26506340

  2. Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush).

    PubMed

    Zhuo, L; Sajdak, S L; Phillips, R B

    1994-08-01

    Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5' external spacer region (5' ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5' ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3-6 kb from the 18S. Sequencing of a 609-b segment of the 5' ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5' ETS using the polymerase chain reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Bat white-nose syndrome: A real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans

    Treesearch

    Laura K Muller; Jeffrey M. Lorch; Daniel L. Lindner; Michael O' Connor; Andrea Gargas; David S. Blehert

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The...

  4. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  5. Escherichia coli σ70 senses sequence and conformation of the promoter spacer region

    PubMed Central

    Singh, Shivani S.; Typas, Athanasios; Hengge, Regine; Grainger, David C.

    2011-01-01

    In bacteria, promoter identification by RNA polymerase is mediated by a dissociable σ factor. The housekeeping σ70 factor of Escherichia coli recognizes two well characterized DNA sequence elements, known as the ‘−10’ and ‘−35’ hexamers. These elements are separated by ‘spacer’ DNA, the sequence of which is generally considered unimportant. Here, we use a combination of bioinformatics, genetics and biochemistry to show that σ70 can sense the sequence and conformation of the promoter spacer region. Our data illustrate how alterations in spacer region sequence can increase promoter activity. This stimulatory effect requires σ70 side chain R451, which is located in close proximity to the non-template strand at promoter position −18. Conversely, R451 is not required to mediate transcriptional stimulation by improvement of the −10 element. Mutation of σ70 residue R451, which is highly conserved, results in reduced growth rate, consistent with a central role in promoter recognition. PMID:21398630

  6. Identification of Swertia mussotii and its adulterant Swertia species by 5S rRNA gene spacer.

    PubMed

    Yu, Man-Tang; Wong, Ka-Lok; Zong, Yu-Ying; Shaw, Pang-Chui; Che, Chun-Tao

    2008-03-01

    This research focused on analyzing the differences of 5S rRNA gene spacer sequences on Swertia mussotii and its commonly used adulterants, including S. franchetiana, S. wolfangiana and S. chirayita. DNA was extracted from the collected Swertia samples. 5S rRNA intergenic spacers were amplified by PCR, sequenced and analyzed. 5S rRNA gene spacer sequences were different between S. mussotii and its other three adulterants. Sequence divergence among species ranged from 30.6% to 65.0%. 5S rRNA spacers may be used as molecular authentication markers to differentiate S. mussotii and other commonly used Swertia adulterants. This result provides reliable and simple reference for the authentication of Swertia genus species.

  7. Intragenomic Variation in the Internal Transcribed Spacer 1 Region of Dientamoeba fragilis as a Molecular Epidemiological Marker▿

    PubMed Central

    Bart, Aldert; van der Heijden, Harold M.; Greve, Sophie; Speijer, Dave; Landman, Wil J.; van Gool, Tom

    2008-01-01

    Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis, a protozoan parasite of birds and a close relative of D. fragilis. This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates,. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis. Within families, it was shown that members can be infected with the same or different strains of D. fragilis. In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite. PMID:18650356

  8. Characterization of mitochondrial control region, two intergenic spacers and tRNAs of Zaprionus indianus (Diptera: Drosophilidae).

    PubMed

    da Silva, Norma Machado; de Souza Dias, Aline; da Silva Valente, Vera Lúcia; Valiati, Victor Hugo

    2009-12-01

    The control region in insects is the major noncoding region in animal mitochondrial DNA (mtDNA), and is responsible for a large part of the variation in the DNA sequence and size of the genome of this organelle. In this study, the mtDNA control region, two intergenic spacers and tRNA genes of a Zaprionus indianus strain were cloned, sequenced and compared with other Drosophila species. The overall A+T content in the Z. indianus control region is 94.3%, and a comparison with other Drosophila species demonstrated that the most conserved region appears to be the 420 base pairs nearest to the tRNA(ile), similar to the findings of other authors. We also describe conserved sequence blocks, including a poly-T involved in the replication process of Drosophila mtDNA; a putative secondary structure also involved in the replication process and repeated sequences. tRNA(ile) sequence demonstrated the greatest variability when the tRNA sequences of species were compared.

  9. High initiation rates at the ribosomal gene promoter do not depend upon spacer transcription.

    PubMed Central

    Labhart, P; Reeder, R H

    1989-01-01

    We report experiments that test the model that in Xenopus laevis, RNA polymerase I is "handed over" in a conservative fashion from the T3 terminator to the adjacent gene promoter. We have introduced transcription-terminating lesions into the ribosomal DNA repeat by irradiating cultured cells with ultraviolet light. We used isolated nuclei to measure the effect of such lesions on transcription. UV damage sufficient to prevent all elongating RNA polymerase from reaching T3 from upstream had no adverse effect on the density of RNA polymerase at the very 5' end of the gene. We conclude that high rates of transcription initiation at the gene promoter do not depend upon polymerase passing from one repeat to the next or on polymerase initiating at the spacer promoters. Images PMID:2470092

  10. Conservation in the first internal transcribed spacer (ITS1) region of Hematodinium perezi (genotype III) from Callinectes sapidus .

    PubMed

    Pagenkopp Lohan, Katrina M; Small, Hamish J; Shields, Jeffrey D; Place, Allen R; Reece, Kimberly S

    2013-03-13

    Hematodinium spp. infections have been reported from blue crabs Callinectes sapidus in high-salinity waters of the USA from New Jersey to Texas. Recently, H. perezi (genotype III) has been proposed as the parasite species and genotype infecting blue crabs from Virginia; however, it is unknown whether this same genotype is present in blue crabs from other locations. To address this question, we collected 317 blue crabs from Massachusetts, Virginia, Georgia, Florida, Louisiana, and Texas to test for the presence of H. perezi (III) using a specific PCR assay targeting the first internal transcribed spacer (ITS1) region of the ribosomal RNA gene complex. To examine the genetic variation within H. perezi (III), ITS1 region sequences from the parasite in blue crabs from multiple locations were compared to each other and to those of H. perezi (III) found in alternate hosts from Virginia. In total, 34 distinct ITS1 sequence variants of the parasite were identified from blue crabs alone, and 38 distinct variants were identified when alternate hosts were included. However, a single ITS1 sequence variant appeared in all geographic regions and hosts, and also in blue crabs sampled from a previous study. The high similarity among all the ITS1 region sequences examined (>98%) and the observation of a single variant found throughout a large geographic range, strongly suggests that a single species and genotype of Hematodinium, specifically H. perezi (III), infects blue crabs from Virginia to Texas and multiple alternate host species in Virginia.

  11. Multigene family of ribosomal DNA in Drosophila melanogaster reveals contrasting patterns of homogenization for IGS and ITS spacer regions. A possible mechanism to resolve this paradox.

    PubMed Central

    Polanco, C; González, A I; de la Fuente; Dover, G A

    1998-01-01

    The multigene family of rDNA in Drosophila reveals high levels of within-species homogeneity and between-species diversity. This pattern of mutation distribution is known as concerted evolution and is considered to be due to a variety of genomic mechanisms of turnover (e.g., unequal crossing over and gene conversion) that underpin the process of molecular drive. The dynamics of spread of mutant repeats through a gene family, and ultimately through a sexual population, depends on the differences in rates of turnover within and between chromosomes. Our extensive molecular analysis of the intergenic spacer (IGS) and internal transcribed spacer (ITS) spacer regions within repetitive rDNA units, drawn from the same individuals in 10 natural populations of Drosophila melanogaster collected along a latitudinal cline on the east coast of Australia, indicates a relatively fast rate of X-Y and X-X interchromosomal exchanges of IGS length variants in agreement with a multilineage model of homogenization. In contrast, an X chromosome-restricted 24-bp deletion in the ITS spacers is indicative of the absence of X-Y chromosome exchanges for this region that is part of the same repetitive rDNA units. Hence, a single lineage model of homogenization, coupled to drift and/or selection, seems to be responsible for ITS concerted evolution. A single-stranded exchange mechanism is proposed to resolve this paradox, based on the role of the IGS region in meiotic pairing between X and Y chromosomes in D. melanogaster. PMID:9584100

  12. Analysis of 16S-23S rRNA internal transcribed spacer regions in Pasteurellaceae isolated from laboratory rodents.

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Christensen, Henrik; Sager, Martin

    2012-09-01

    The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, "Hemophilus influenzaemurium" and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species-specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of "H. influenzaemurium", a larger ITS(ile+ala) which contained the genes for tRNA(Ile(GAU)) and tRNA(Ala(UGC)) and a smaller ITS(glu) with the tRNA(Glu(UUC)) gene. "H. influenzaemurium" revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITS(ile+ala) and 56.5 to 86.8% for ITS(glu). Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA

    PubMed Central

    Li, Dezhu; Sun, Yongzhen; Niu, Yunyun; Chen, Zhiduan; Luo, Hongmei; Pang, Xiaohui; Sun, Zhiying; Liu, Chang; Lv, Aiping; Deng, Youping; Larson-Rabin, Zachary; Wilkinson, Mike; Chen, Shilin

    2012-01-01

    Background Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. Methodology/Principal Findings In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. Conclusions Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification. PMID:22952830

  14. DNA authentication of Plantago Herb based on nucleotide sequences of 18S-28S rRNA internal transcribed spacer region.

    PubMed

    Sahin, Fatma Pinar; Yamashita, Hiromi; Guo, Yahong; Terasaka, Kazuyoshi; Kondo, Toshiya; Yamamoto, Yutaka; Shimada, Hiroshi; Fujita, Masao; Kawasaki, Takeshi; Sakai, Eiji; Tanaka, Toshihiro; Goda, Yukihiro; Mizukami, Hajime

    2007-07-01

    Internal transcribed spacer (ITS) regions of nuclear ribosomal RNA gene were amplified from 23 plant- and herbarium specimens belonging to eight Plantago species (P. asiatica, P. depressa, P. major, P. erosa, P. hostifolia, P. camtschatica, P. virginica and P. lanceolata). Sequence comparison indicated that these Plantago species could be identified based on the sequence type of the ITS locus. Sequence analysis of the ITS regions amplified from the crude drug Plantago Herb obtained in the markets indicated that all the drugs from Japan were derived from P. asiatica whereas the samples obtained in China were originated from various Plantago species including P. asiatica, P. depressa, P. major and P. erosa.

  15. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    PubMed

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions. Copyright © 2012 Elsevier Inc

  16. Insertions or Deletions (Indels) in the rrn 16S-23S rRNA Gene Internal Transcribed Spacer Region (ITS) Compromise the Typing and Identification of Strains within the Acinetobacter calcoaceticus-baumannii (Acb) Complex and Closely Related Members

    PubMed Central

    Maslunka, Christopher; Gifford, Bianca; Tucci, Joseph; Gürtler, Volker; Seviour, Robert J.

    2014-01-01

    To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2–13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species. PMID:25141005

  17. Impact of Asymmetric Dual-k Spacer in the Underlap Regions of Sub 20 nm NMOSFET with Gate Stack

    NASA Astrophysics Data System (ADS)

    Chakraborty, Shramana; Dasgupta, Arpan; Das, Rahul; Kundu, Atanu; Sarkar, Chandan K.

    2016-10-01

    This paper shows the systematic study of underlap double gate (U-DG) NMOSFETs with Gate Stack (GS) under the influence of dual-k spacers at the different underlap regions. In highly scaled devices, underlap is used at the Source and Drain side so as to reduce the short channel effects (SCE's) but at the cost of low on current (ION) and increased channel resistance. The high-k spacers are used to counter this problem. The ION is improved but at the cost of highly enhanced parasitic capacitances. This paper explores the possibility of using asymmetric dual-k spacer at the source underlap side so as to counter the shortcomings of high-k spacers in highly scaled devices on the basis of analog parameters: ION, gm, gm/ID, and intrinsic gain, gmRo and RF performance in terms of parasitic gate capacitance (Cgs, Cgd and Cgg),gate to source/drain resistances (Rgs and Rgd), transport delay (τm), the unity current gain cut-off frequency (fT) and the maximum frequency of oscillation (fmax). A single stage amplifier performance is also analyzed where it has been seen that the asymmetric dual-k spacer at the source underlap side gives better performance as compared to the other devices under comparison.

  18. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

    Treesearch

    Daniel L. Lindner; Tor Carlsen; Henrik Nilsson; Marie Davey; Trond Schumacher; Havard. Kauserud

    2013-01-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular...

  19. Molecular phylogenetic analysis of Indonesia Solanaceae based on DNA sequences of internal transcribed spacer region

    NASA Astrophysics Data System (ADS)

    Hidayat, Topik; Priyandoko, Didik; Islami, Dina Karina; Wardiny, Putri Yunitha

    2016-02-01

    Solanaceae is one of largest family in Angiosperm group with highly diverse in morphological character. In Indonesia, this group of plant is very popular due to its usefulness as food, ornamental and medicinal plants. However, investigation on phylogenetic relationship among the member of this family in Indonesia remains less attention. The purpose of this study was to evaluate the phylogenetics relationship of the family especially distributed in Indonesia. DNA sequences of Internal Transcribed Spacer (ITS) region of 19 species of Solanaceae and three species of outgroup, which belongs to family Convolvulaceae, Apocynaceae, and Plantaginaceae, were isolated, amplified, and sequenced. Phylogenetic tree analysis based on parsimony method was conducted with using data derived from the ITS-1, 5.8S, and ITS-2, separately, and the combination of all. Results indicated that the phylogenetic tree derived from the combined data established better pattern of relationship than separate data. Thus, three major groups were revealed. Group 1 consists of tribe Datureae, Cestreae, and Petunieae, whereas group 2 is member of tribe Physaleae. Group 3 belongs to tribe Solaneae. The use of the ITS region as a molecular markers, in general, support the global Solanaceae relationship that has been previously reported.

  20. Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout

    USGS Publications Warehouse

    Rasmussen, C.; Purcell, M.K.; Gregg, J.L.; LaPatra, S.E.; Winton, J.R.; Hershberger, P.K.

    2010-01-01

    The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of ~740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

  1. [Discrimination of psychoactive fungi (commonly called "magic mushrooms") based on the DNA sequence of the internal transcribed spacer region].

    PubMed

    Maruyama, Takuro; Shirota, Osamu; Kawahara, Nobuo; Yokoyama, Kazumasa; Makino, Yukiko; Goda, Yukihiro

    2003-02-01

    'Magic mushrooms' (MMs) are psychoactive fungi containing the hallucinogenic compounds, psilocin (1) and psilocybin (2). Since June 6, 2002, these fungi have been regulated by the Narcotics and Psychotropics Control Law in Japan. Because there are many kinds of MMs and they are sold even as dry powders in local markets, it is very difficult to identify the original species of the MMs by morphological observation. Therefore, we investigated the internal transcribed spacer (ITS) region in the ribosomal RNA gene of MMs obtained in Japanese markets to classify them by a genetic approach. Based on the size and nucleotide sequence of the ITS region amplified by PCR, tested MMs were classified into 6 groups. Furthermore, a comparison of the DNA sequences of the MMs with those of authentic samples or with those found in the databases (GenBank, EMBL and DDBJ) made it possible to identify the species of tested MMs. Analysis by LC revealed that psilocin (1) was contained at the highest level in Panaeolus cyanescens among the MMs, but was absent in the Amanita species.

  2. Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region.

    PubMed

    Booton, G C; Kaufman, L; Chandler, M; Oguto-Ohwayo, R; Duan, W; Fuerst, P A

    1999-03-01

    The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.

  3. Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran.

    PubMed

    Ghatee, Mohammad Amin; Sharifi, Iraj; Kuhls, Katrin; Kanannejad, Zahra; Harandi, Majid Fasihi; de Almeida, Marcos E; Hatam, Gholamreza; Mirhendi, Hossein

    2014-09-01

    Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica.

  4. Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure.

    PubMed

    Osorio, Carlos R; Collins, Matthew D; Romalde, Jesús L; Toranzo, Alicia E

    2005-02-01

    Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.

  5. Molecular analysis of fungal populations in patients with oral candidiasis using internal transcribed spacer region.

    PubMed

    Ieda, Shinsuke; Moriyama, Masafumi; Takeshita, Toru; Takashita, Toru; Maehara, Takashi; Imabayashi, Yumi; Shinozaki, Shoichi; Tanaka, Akihiko; Hayashida, Jun-Nosuke; Furukawa, Sachiko; Ohta, Miho; Yamashita, Yoshihisa; Nakamura, Seiji

    2014-01-01

    Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection.

  6. Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region

    PubMed Central

    Ieda, Shinsuke; Moriyama, Masafumi; Takashita, Toru; Maehara, Takashi; Imabayashi, Yumi; Shinozaki, Shoichi; Tanaka, Akihiko; Hayashida, Jun-Nosuke; Furukawa, Sachiko; Ohta, Miho; Yamashita, Yoshihisa; Nakamura, Seiji

    2014-01-01

    Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection. PMID:24979710

  7. RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.

    PubMed

    Volokhov, Dmitriy V; Simonyan, Vahan; Davidson, Maureen K; Chizhikov, Vladimir E

    2012-01-01

    Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in

  8. PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

    PubMed Central

    2010-01-01

    Background The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species. Results In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. Conclusion This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations

  9. Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus: implications for molecular diagnosis in clinical microbiology laboratories.

    PubMed

    Woo, Patrick C Y; Leung, Shui-Yee; To, Kelvin K W; Chan, Jasper F W; Ngan, Antonio H Y; Cheng, Vincent C C; Lau, Susanna K P; Yuen, Kwok-Yung

    2010-01-01

    Although internal transcribed spacer region (ITS) sequence heterogeneity has been reported in a few fungal species, it has very rarely been reported in pathogenic fungi and has never been described in Mucorales, causes of the highly fatal mucormycosis. In a recent outbreak investigation of intestinal mucormycosis due to Rhizopus microsporus infection in patients with hematological malignancies, PCR of the ITS of four of the 28 R. microsporus strains, P11, P12, D3-1, and D4-1, showed thick bands at about 700 bp. Direct sequencing of the purified bands showed frequent double peaks along all of the sequence traces and occasional triple peaks for P12, D3-1, and D4-1. The thick bands of the four R. microsporus strains were purified and cloned. Sequencing of 10 clones for each strain revealed two different ITS sequences for P11 and three different ITS sequences for P12, D3-1, and D4-1. Variations in ITS sequence among the different ribosomal DNA (rDNA) operons in the same strain were observed in only ITS1 and ITS2 and not the 5.8S rDNA region. One copy of P11, P12, and D4-1, respectively, and one copy of P11, P12, D3-1, and D4-1, respectively, showed identical sequences. This represents the first evidence of ITS sequence heterogeneity in Mucorales. ITS sequence heterogeneity is an obstacle to molecular identification and genotyping of fungi in clinical microbiology laboratories. When thick bands and double peaks are observed during PCR sequencing of a gene target, such a strain should be sent to reference laboratories proficient in molecular technologies for further identification and/or genotyping.

  10. Identification of fungi based on the nucleotide sequence homology of their internal transcribed spacer 1 (ITS1) region.

    PubMed

    Narutaki, Shoji; Takatori, Kosuke; Nishimura, Hidekatsu; Terashima, Hiroshi; Sasaki, Tsuguo

    2002-01-01

    In this study, we examined the identification of fungi based on the sequence homology of the internal transcribed spacer 1 (ITS1) region. A newly designed primer pair could amplify the target region of all 42 strains tested. The PCR products were sequenced and the sequence homologies were searched by BLAST. It was demonstrated that this method is a reliable identification method at the genus or species level. At present, available databases are still insufficient to identify some fungi, but with the accumulation of further data in the ITS1 database, this method will be available for the identification of fungi.

  11. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions.

    PubMed

    Scorzetti, Gloria; Fell, J W; Fonseca, A; Statzell-Tallman, Adele

    2002-12-01

    Basidiomycetous yeasts in the Urediniomycetes and Hymenomycetes were examined by sequence analysis in two ribosomal DNA regions: the D1/D2 variable domains at the 5' end of the large subunit rRNA gene (D1/D2) and the internal transcribed spacers (ITS) 1 and 2. Four major lineages were recognized in each class: Microbotryum, Sporidiobolus, Erythrobasidium and Agaricostilbum in the Urediniomycetes; Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales in the Hymenomycetes. Bootstrap support for many of the clades within those lineages is weak; however, phylogenetic analysis provides a focal point for in-depth study of biological relationships. Combined sequence analysis of the D1/D2 and ITS regions is recommended for species identification, while species definition requires classical biological information such as life cycles and phenotypic characterization.

  12. Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum).

    PubMed

    May, C E; Appels, R

    1987-09-01

    Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.

  13. 16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations.

    PubMed

    Callejón, R; Halajian, A; de Rojas, M; Marrugal, A; Guevara, D; Cutillas, C

    2012-05-25

    Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis). Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Diversity of Salmonella Strains Isolated from the Aquatic Environment as Determined by Serotyping and Amplification of the Ribosomal DNA Spacer Regions

    PubMed Central

    Baudart, Julia; Lemarchand, Karine; Brisabois, Anne; Lebaron, Philippe

    2000-01-01

    Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186–194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates. PMID:10742240

  15. A new PCR primer for the identification of Paracoccidioides brasiliensis based on rRNA sequences coding the internal transcribed spacers (ITS) and 5 x 8S regions.

    PubMed

    Imai, T; Sano, A; Mikami, Y; Watanabe, K; Aoki, F H; Branchini, M L; Negroni, R; Nishimura, K; Miyaji, M

    2000-08-01

    Internal transcribed spacer (ITS) genes including the 5.8S ribosomal (r)RNA of Paracoccidioides brasiliensis were amplified and the DNA sequences were determined. Based on a comparison of the sequence information, a new polymerase chain reaction (PCR) primer pair was designed for specific amplification of DNA for P. brasiliensis. This primer pair amplified a 418-bp DNA sequence and was 100% successful in identifying 29 strains of P. brasiliensis (including the reference strains) isolated from the regions of Brazil, Costa Rica, Japan, Argentina or from different sources. The results of specificity tests of these primers to compare the fungus with those of Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei are also reported.

  16. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys.

    PubMed

    Walters, William; Hyde, Embriette R; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A; Jansson, Janet K; Caporaso, J Gregory; Fuhrman, Jed A; Apprill, Amy; Knight, Rob

    2016-01-01

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for

  17. Sequence diversity in the 16S-23S intergenic spacer region (ISR) of the rRNA operons in representatives of the Escherichia coli ECOR collection.

    PubMed

    Antón, A I; Martínez-Murcia, A J; Rodríguez-Valera, F

    1998-07-01

    The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S-23S intergenic spacer region (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for tRNA(Ile-1) and tRNA(Ala-1B). To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.

  18. Identification of Giardia species and Giardia duodenalis assemblages by sequence analysis of the 5.8S rDNA gene and internal transcribed spacers.

    PubMed

    Cacciò, Simone M; Beck, Relja; Almeida, Andre; Bajer, Anna; Pozio, Edoardo

    2010-05-01

    PCR assays have been developed mainly to assist investigations into the epidemiology of Giardia duodenalis, the only species in the Giardia genus having zoonotic potential. However, a reliable identification of all species is of practical importance, particularly when water samples and samples from wild animals are investigated. The aim of the present work was to genotype Giardia species and G. duodenalis assemblages using as a target the region spanning the 5.8S gene and the 2 flanking internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene. Primers were designed to match strongly conserved regions in the 3' end of the small subunit and in the 5' end of the large subunit ribosomal genes. The corresponding region (about 310 bp) was amplified from 49 isolates of both human and animal origin, representing all G. duodenalis assemblages as well as G. muris and G. microti. Sequence comparison and phylogenetic analysis showed that G. ardeae, G. muris, G. microti as well as the 7 G. duodenalis assemblages can be easily distinguished. Since the major subgroups within the zoonotic assemblages A and B can be identified by sequence analysis, this assay is also informative for molecular epidemiological studies.

  19. RNA Replication from the Simian Virus 5 Antigenomic Promoter Requires Three Sequence-Dependent Elements Separated by Sequence-Independent Spacer Regions

    PubMed Central

    Keller, Michael A.; Murphy, Susan K.; Parks, Griffith D.

    2001-01-01

    We have previously shown for the paramyxovirus simian virus 5 (SV5) that a functional promoter for RNA replication requires proper spacing between two discontinuous elements: a 19-base segment at the 3′ terminus (conserved region I [CRI]) and an 18-base internal region (CRII) that is contained within the coding region of the L protein gene. In the work described here, we have used a reverse-genetics system to determine if the 53-base segment between CRI and CRII contains additional sequence-specific signals required for optimal replication or if this segment functions solely as a sequence-independent spacer region. A series of copyback defective interfering minigenome analogs were constructed to contain substitutions of nonviral sequences in place of bases 21 to 72 of the antigenomic promoter, and the relative level of RNA replication was measured by Northern blot analysis. The results from our mutational analysis indicate that in addition to CRI and CRII, optimal replication from the SV5 antigenomic promoter requires a third sequence-dependent element located 51 to 66 bases from the 3′ end of the RNA. Minigenome RNA replication was not affected by changes in the either the position of this element in relation to CRI and CRII or the predicted hexamer phase of NP encapsidation. Thus, optimal RNA replication from the SV5 antigenomic promoter requires three sequence-dependent elements, CRI, CRII and bases 51 to 66. PMID:11264390

  20. Genetic diversity of Histoplasma capsulatum strains isolated from Argentina based on nucleotide sequence variations in the internal transcribed spacer regions of rDNA.

    PubMed

    Landaburu, Fernanda; Cuestas, María Luján; Rubio, Andrea; Elías, Nahuel Alejandro; Daneri, Gabriela Lopez; Veciño, Cecilia; Iovannitti, Cristina A; Mujica, María Teresa

    2014-05-01

    The internal transcribed spacer (ITS) regions of rDNA genes of 49 Histoplasma capsulatum (48 from clinical samples and one from soil) isolates were examined. Nucleotide sequence heterogeneity within this region was useful for phylogenetic classification of H. capsulatum and species identification. Thus, in 45 of 49 isolates we observed higher percentages of identity in the nucleotide sequences of ITS regions when the isolates studied herein were compared with those reported in our country in the South America B clade. Phylogenetic analyses of rDNA sequences corresponding to the 537 bp of the ITS region obtained from H. capsulatum isolates assigned South America type B clade (45 isolates), North America type 1 and Asia clade (2 isolates each one). H. capsulatum strains isolated from soil and from patients living in Argentina (45 of 49) clustered together with the H. capsulatum isolates of the South America B clade. The high level of genetic similarity among our isolates suggests that almost one genetic population is present in the microenvironment. Isolates described as H. capsulatum var. capsulatum or var. farciminosum (2 isolates) did not form a monophyletic group and were found in the Asia clade. Subsequent studies are needed to properly identify these isolates.

  1. DNA polymorphism in morels: complete sequences of the internal transcribed spacer of genes coding for rRNA in Morchella esculenta (yellow morel) and Morchella conica (black morel).

    PubMed

    Wipf, D; Munch, J C; Botton, B; Buscot, F

    1996-09-01

    The internal transcribed spacer (ITS) of the gene coding for rRNA was sequenced in both directions with the gene walking technique in a black morel (Morchella conica) and a yellow morel (M. esculenta) to elucidate the ITS length discrepancy between the two species groups (750-bp ITS in black morels and 1,150-bp ITS in yellow morels.

  2. An AluI fragment isolated from lake trout (Salvelinus namaycush), maps to the intergenic spacer region of the rDNA cistron.

    PubMed

    Reed, K M; Oakley, T H; Phillips, R B

    1997-02-20

    The relationship between a 217-bp AluI fragment (SnAluI-33c) from lake trout (Salvelinus namaycush) which hybridizes to the nucleolar organizer regions (NORs) and the ribosomal RNA genes was examined by Southern analysis and comparative hybridization. Restriction enzymes with recognition sites mapped in the lake trout rDNA cistron were used to digest genomic DNA into fragments of predetermined size. Comparison of the hybridization pattern of SnAluI-33c with those of two rDNA-specific probes placed this fragment within the intergenic spacer region of the rDNA cistron, approximately 3 kb upstream (5') of the 18S gene. This finding is consistent with in situ hybridization experiments showing hybridization of this fragment to sites of rDNA [Reed, K.M. and Phillips, R.B., Cytogenet. Cell Genet. 70 (1995) 104-107]. Based on cross hybridization and sequence comparisons, homologous sequences are present in other salmonid species.

  3. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi.

    PubMed

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-06-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3-5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

  4. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

    PubMed Central

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-01-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3–5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions. PMID:23789083

  5. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    USDA-ARS?s Scientific Manuscript database

    Six DNA regions were evaluated in a multi-national, multi-laboratory consortium as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it...

  6. Molecular phylogeny of tribe Forsythieae (Oleaceae) based on nuclear ribosomal DNA internal transcribed spacers and plastid DNA trnL-F and matK gene sequences.

    PubMed

    Kim, Dong-Kap; Kim, Joo-Hwan

    2011-05-01

    The tribe Forsythieae comprises 2 genera (Forsythia and Abeliophyllum) and 14 species distributed mostly in the Far East. Although Forsythieae is considered monophyletic, with many symplesiomorphic characters, the phylogenetic status of Abeliophyllum remains controversial. We assessed the phylogenetic relationships of Forsythieae, based on a 3.3-kb plastid fragment (trnL-F region and matK gene) and nuclear internal transcribed spacer (ITS) region DNA sequences. We obtained a highly resolved and strongly supported topology with possible outgroups. The topology of the combined tree was congruent with those of the ITS region and matK gene. Maximum parsimony, maximum likelihood, and Bayesian inference tree analyses for the combined data also yielded identical relationships. Combined sequence data strongly supported the monophyly of Forsythieae and the close relationship between Fontanesia and Jasminum. Oleaceae, not Fontanesia, was found to be a sister group to Forsythieae. Moreover, the genus Abeliophyllum was distinctly independent of Forsythia. Three Forsythia lineages were suggested: (a) ONJ (ovata-nakaii-japonica clade), (b) VGE (viridissima-giraldiana-europaea), and (c) KISS (koreana-intermedia-saxatilis-suspensa). Our results indicated that F. × intermedia is not a hybrid between F. suspensa and F. viridissima, but further studies are needed to determine its taxonomic identity. Furthermore, the diverse fruit shapes in Oleaceae are assumed to be the result of parallelism or convergence.

  7. [Comparative characteristics of intergenic spacers of ribosomal RNA gene cluster in mosquitoes of the genus Culex (Diptera: Culicidae)].

    PubMed

    Shaĭkevich, E V; Zagoskin, M V; Mukha, D V

    2013-01-01

    Nucleotide sequences of intergenic spacer of ribosomal RNA gene cluster (rIGS) were identified in mosquitoes Culex modestus, Culex torrentium and Culex pipiens pallens. The level of interpopulation variability of the rIGS in the subspecies C. pipiens pipiens (form pipiens--mosquitoes that inhabit the open waters, and form molestus--mosquitoes that inhabit basements) living in Russia was estimated. No extensive repetitive sequences characteristic of the rIGS of all previously described species of mosquitoes were found within the rIGS of Culex mosquitoes. At the same time, evolutionarily conserved motifs and relatively short degenerate sequences of different classes of transposable elements, as well as multiple blocks of variable microsatellite repeats were identified. Our data demonstrated that the rIGS of Culex mosquitoes can be considered as a promising molecular marker for the analysis of population and phylogenetic relationships within this group of insects.

  8. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    SciTech Connect

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob; Bik, Holly

    2015-12-22

    ABSTRACT

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection ofThaumarchaeotaand clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

    ImportanceWe continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to

  9. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    PubMed Central

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy

    2015-01-01

    ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers

  10. Evolution of the rpoB-psbZ region in fern plastid genomes: notable structural rearrangements and highly variable intergenic spacers

    PubMed Central

    2011-01-01

    Background The rpoB-psbZ (BZ) region of some fern plastid genomes (plastomes) has been noted to go through considerable genomic changes. Unraveling its evolutionary dynamics across all fern lineages will lead to clarify the fundamental process shaping fern plastome structure and organization. Results A total of 24 fern BZ sequences were investigated with taxon sampling covering all the extant fern orders. We found that: (i) a tree fern Plagiogyria japonica contained a novel gene order that can be generated from either the ancestral Angiopteris type or the derived Adiantum type via a single inversion; (ii) the trnY-trnE intergenic spacer (IGS) of the filmy fern Vandenboschia radicans was expanded 3-fold due to the tandem 27-bp repeats which showed strong sequence similarity with the anticodon domain of trnY; (iii) the trnY-trnE IGSs of two horsetail ferns Equisetum ramosissimum and E. arvense underwent an unprecedented 5-kb long expansion, more than a quarter of which was consisted of a single type of direct repeats also relevant to the trnY anticodon domain; and (iv) ycf66 has independently lost at least four times in ferns. Conclusions Our results provided fresh insights into the evolutionary process of fern BZ regions. The intermediate BZ gene order was not detected, supporting that the Adiantum type was generated by two inversions occurring in pairs. The occurrence of Vandenboschia 27-bp repeats represents the first evidence of partial tRNA gene duplication in fern plastomes. Repeats potentially forming a stem-loop structure play major roles in the expansion of the trnY-trnE IGS. PMID:21486489

  11. Evolution of the rpoB-psbZ region in fern plastid genomes: notable structural rearrangements and highly variable intergenic spacers.

    PubMed

    Gao, Lei; Zhou, Yuan; Wang, Zhi-Wei; Su, Ying-Juan; Wang, Ting

    2011-04-13

    The rpoB-psbZ (BZ) region of some fern plastid genomes (plastomes) has been noted to go through considerable genomic changes. Unraveling its evolutionary dynamics across all fern lineages will lead to clarify the fundamental process shaping fern plastome structure and organization. A total of 24 fern BZ sequences were investigated with taxon sampling covering all the extant fern orders. We found that: (i) a tree fern Plagiogyria japonica contained a novel gene order that can be generated from either the ancestral Angiopteris type or the derived Adiantum type via a single inversion; (ii) the trnY-trnE intergenic spacer (IGS) of the filmy fern Vandenboschia radicans was expanded 3-fold due to the tandem 27-bp repeats which showed strong sequence similarity with the anticodon domain of trnY; (iii) the trnY-trnE IGSs of two horsetail ferns Equisetum ramosissimum and E. arvense underwent an unprecedented 5-kb long expansion, more than a quarter of which was consisted of a single type of direct repeats also relevant to the trnY anticodon domain; and (iv) ycf66 has independently lost at least four times in ferns. Our results provided fresh insights into the evolutionary process of fern BZ regions. The intermediate BZ gene order was not detected, supporting that the Adiantum type was generated by two inversions occurring in pairs. The occurrence of Vandenboschia 27-bp repeats represents the first evidence of partial tRNA gene duplication in fern plastomes. Repeats potentially forming a stem-loop structure play major roles in the expansion of the trnY-trnE IGS.

  12. Structural, biocomplexation and gene delivery properties of hydroxyethylated gemini surfactants with varied spacer length.

    PubMed

    Zakharova, Lucia Ya; Gabdrakhmanov, Dinar R; Ibragimova, Alsu R; Vasilieva, Elmira A; Nizameev, Irek R; Kadirov, Marsil K; Ermakova, Elena A; Gogoleva, Natalia E; Faizullin, Dzhigangir A; Pokrovsky, Andrey G; Korobeynikov, Vladislav A; Cheresiz, Sergey V; Zuev, Yuriy F

    2016-04-01

    Gemini surfactants with hexadecyl tails and hydroxyethylated head groups bridged with tetramethylene (G4), hexamethylene (G6) and dodecamethylene (G12) spacers were shown to self-assemble at the lower critical micelle concentration compared to their conventional m-s-m analogs. The lipoplex formation and the plasmid DNA transfer into different kinds of host cells were studied. In the case of eukaryotic cells, high transfection efficacy has been demonstrated for DNA-gemini complexes, which increased as follows: G6G4>G12 has been obtained in the case of transformation of bacterial cells with plasmid DNA-gemini complexes, mediated by electroporation technique. Solely G6 shows transformation efficacy exceeding the control result (uncomplexed DNA), while the inhibitory effect occurs for G4 and G12. Analysis of physico-chemical features of single surfactants and lipoplexes shows that compaction and condensation effects change as follows: G6

  13. Phylogenetics of Bonamia parasites based on small subunit and internal transcribed spacer region ribosomal DNA sequence data.

    PubMed

    Hill, Kristina M; Stokes, Nancy A; Webb, Stephen C; Hine, P Mike; Kroeck, Marina A; Moore, James D; Morley, Margaret S; Reece, Kimberly S; Burreson, Eugene M; Carnegie, Ryan B

    2014-07-24

    The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

  14. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans.

    PubMed

    Muller, Laura K; Lorch, Jeffrey M; Lindner, Daniel L; O'Connor, Michael; Gargas, Andrea; Blehert, David S

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was qualified further by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

  15. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

    USGS Publications Warehouse

    Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

  16. Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S-23S rRNA internal transcribed spacer regions.

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Sager, Martin

    2013-08-01

    [Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Genetic diversity of two Daphnia-infecting microsporidian parasites, based on sequence variation in the internal transcribed spacer region.

    PubMed

    González-Tortuero, Enrique; Rusek, Jakub; Maayan, Inbar; Petrusek, Adam; Piálek, Lubomír; Laurent, Stefan; Wolinska, Justyna

    2016-05-20

    Microsporidia are spore-forming obligate intracellular parasites that include both emerging pathogens and economically important disease agents. However, little is known about the genetic diversity of microsporidia. Here, we investigated patterns of geographic population structure, intraspecific genetic variation, and recombination in two microsporidian taxa that commonly infect cladocerans of the Daphnia longispina complex in central Europe. Taken together, this information helps elucidate the reproductive mode and life-cycles of these parasite species. Microsporidia-infected Daphnia were sampled from seven drinking water reservoirs in the Czech Republic. Two microsporidia species (Berwaldia schaefernai and microsporidium lineage MIC1) were sequenced at the internal transcribed spacer (ITS) region, using the 454 pyrosequencing platform. Geographical structure analyses were performed applying Fisher's exact tests, analyses of molecular variance, and permutational MANOVA. To evaluate the genetic diversity of the ITS region, the number of polymorphic sites and Tajima's and Watterson's estimators of theta were calculated. Tajima's D was also used to determine if the ITS in these taxa evolved neutrally. Finally, neighbour similarity score and pairwise homology index tests were performed to detect recombination events. While there was little variation among Berwaldia parasite strains infecting different host populations, the among-population genetic variation of MIC1 was significant. Likewise, ITS genetic diversity was lower in Berwaldia than in MIC1. Recombination signals were detected only in Berwaldia. Genetic tests showed that parasite populations could have expanded recently after a bottleneck or that the ITS could be under negative selection in both microsporidia species. Recombination analyses might indicate cryptic sex in Berwaldia and pure asexuality in MIC1. The differences observed between the two microsporidian species present an exciting opportunity to

  18. Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers.

    PubMed

    de Llanos Frutos, Rosa; Fernández-Espinar, M Teresa; Querol, Amparo

    2004-04-01

    The PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene is applied to the identification of yeasts belonging to the genus Candida. This methodology has previously been used for the identification of some species of this genus, but in the present work this application has been applied to the identification and characterisation of a greater number of species of the genus Candida, with a special survey of species of clinical and biotechnological interest. Among the species of the genus Candida, the high variability observed, both in the length of the amplified region (ranging between 390 and 900 bp) and in their restriction patterns, allows the unequivocal identification to the species level, with the exception of the group of species that comprises C. membranifaciens, C. conglobata, C. atlantica, C. atmosphaerica, and C. oleophila, that required the sequencing of the D1/D2 domain of the 26S rRNA gene or the 5.8S-ITS region for their proper differentiation. The 5.8S-ITS restriction analysis also failed in the differentiation of species within the pairs C.aaseri/C.butyri,C.fructus/C.musae,C.santamariae var. santamariae / C. beechii and C. zeylanoides / C. krissii. In this case, the high sequence similarities obtained for their 26S D1/D2 domain and the 5.8S-ITS region indicate that each pair of species should be considered as a single species. The main purpose of this work is to generate a database for a high number of yeast species, of both biotechnological and clinical interest, and to facilitate their easy, fast, and reliable identification. The present work improves the database available online at the IATA web page (http://motor.edinfo.es/iata/) with the patterns of 75 species belonging to the genus Candida.

  19. How does the spacer length of cationic gemini lipids influence the lipoplex formation with plasmid DNA? Physicochemical and biochemical characterizations and their relevance in gene therapy.

    PubMed

    Muñoz-Úbeda, Mónica; Misra, Santosh K; Barrán-Berdón, Ana L; Datta, Sougata; Aicart-Ramos, Clara; Castro-Hartmann, Pablo; Kondaiah, Paturu; Junquera, Elena; Bhattacharya, Santanu; Aicart, Emilio

    2012-12-10

    Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n=2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA−, a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA−=−2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEMand SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out

  20. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

  1. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed Central

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola. PMID:8979356

  2. Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts.

    PubMed

    Varehese, Rajani; Chauhan, Vineeta S; Misra, Arvind K

    2003-06-01

    While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.

  3. Molecular organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural features and evolution of piscine 5S rRNA genes and nontranscribed intergenic spacers.

    PubMed

    Pasolini, Paola; Costagliola, Domenico; Rocco, Lucia; Tinti, Fausto

    2006-05-01

    The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes.

  4. Ribosomal RNA gene silencing in interpopulation hybrids of Tigriopus californicus: nucleolar dominance in the absence of intergenic spacer subrepeats.

    PubMed

    Flowers, Jonathan M; Burton, Ronald S

    2006-07-01

    A common feature of interspecific animal and plant hybrids is the uniparental silencing of ribosomal RNA gene transcription, or nucleolar dominance. A leading explanation for the genetic basis of nucleolar dominance in animal hybrids is the enhancer-imbalance model. The model proposes that limiting transcription factors are titrated by a greater number of enhancer-bearing subrepeat elements in the intergenic spacer (IGS) of the dominant cluster of genes. The importance of subrepeats for nucleolar dominance has repeatedly been supported in competition assays between Xenopus laevis and X. borealis minigene constructs injected into oocytes. However, a more general test of the importance of IGS subrepeats for nuclear dominance in vivo has not been conducted. In this report, rRNA gene expression was examined in interpopulation hybrids of the marine copepod Tigriopus californicus. This species offers a rare opportunity to test the role of IGS subrepeats in nucleolar dominance because the internal subrepeat structure, found in the IGS of virtually all animal and plant species, is absent in T. californicus. Our results clearly establish that nucleolar dominance occurs in F1 and F2 interpopulation hybrids of this species. In the F2 generation, nucleolar dominance appears to break down in some hybrids in a fashion that is inconsistent with a transcription factor titration model. These results are significant because they indicate that nucleolar dominance can be established and maintained without enhancer-bearing repeat elements in the IGS. This challenges the generality of the enhancer-imbalance model for nucleolar dominance and suggests that dominance of rRNA transcription in animals may be determined by epigenetic factors as has been established in plants.

  5. Ribosomal RNA Gene Silencing in Interpopulation Hybrids of Tigriopus californicus: Nucleolar Dominance in the Absence of Intergenic Spacer Subrepeats

    PubMed Central

    Flowers, Jonathan M.; Burton, Ronald S.

    2006-01-01

    A common feature of interspecific animal and plant hybrids is the uniparental silencing of ribosomal RNA gene transcription, or nucleolar dominance. A leading explanation for the genetic basis of nucleolar dominance in animal hybrids is the enhancer-imbalance model. The model proposes that limiting transcription factors are titrated by a greater number of enhancer-bearing subrepeat elements in the intergenic spacer (IGS) of the dominant cluster of genes. The importance of subrepeats for nucleolar dominance has repeatedly been supported in competition assays between Xenopus laevis and X. borealis minigene constructs injected into oocytes. However, a more general test of the importance of IGS subrepeats for nuclear dominance in vivo has not been conducted. In this report, rRNA gene expression was examined in interpopulation hybrids of the marine copepod Tigriopus californicus. This species offers a rare opportunity to test the role of IGS subrepeats in nucleolar dominance because the internal subrepeat structure, found in the IGS of virtually all animal and plant species, is absent in T. californicus. Our results clearly establish that nucleolar dominance occurs in F1 and F2 interpopulation hybrids of this species. In the F2 generation, nucleolar dominance appears to break down in some hybrids in a fashion that is inconsistent with a transcription factor titration model. These results are significant because they indicate that nucleolar dominance can be established and maintained without enhancer-bearing repeat elements in the IGS. This challenges the generality of the enhancer-imbalance model for nucleolar dominance and suggests that dominance of rRNA transcription in animals may be determined by epigenetic factors as has been established in plants. PMID:16648582

  6. Phylogenetic relationships in the Gesnerioideae (Gesneriaceae) based on nrDNA ITS and cpDNA trnL-F and trnE-T spacer region sequences.

    PubMed

    Zimmer, Elizabeth A; Roalson, Eric H; Skog, Laurence E; Boggan, John K; Idnurm, Alexander

    2002-02-01

    The Gesnerioideae includes most of the New World members of the Gesneriaceae family and is currently considered to include five tribes: Beslerieae, Episcieae, Gesnerieae, Gloxinieae, and Napeantheae. This study presents maximum parsimony and maximum likelihood phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer regions (ITS), and the chloroplast DNA trnL intron, trnL-trnF intergenic spacer region, and trnE-trnT intergenic spacer region sequences. The ITS and cpDNA data sets strongly support the monophyly of a Beslerieae/Napeantheae clade; an Episcieae clade; a Gesnerieae clade; a Gloxinieae clade minus Sinningia, Sinningia relatives, and Gloxinia sarmentiana; and a Sinningia/Paliavana/Vanhouttea clade. This is the first study to provide strong statistical support for these tribes/clades. These analyses suggest that Sinningia and relatives should be considered as a separate tribe. Additionally, generic relationships are explored, including the apparent polyphyly of Gloxinia. Chromosome number changes are minimized on the proposed phylogeny, with the exception of the n = 11 taxa of the Gloxinieae. Scaly rhizomes appear to have been derived once in the Gloxinieae sensu stricto. The number of derivations of the inferior ovary is unclear: either there was one derivation with a reversal to a superior ovary in the Episcieae, or there were multiple independent derivations of the inferior ovary.

  7. Inter- and intraspecific genomic variability of the 16S-23S intergenic spacer regions (ISR) in representatives of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans.

    PubMed

    Ni, Yong-Qing; Yang, Yuan; Bao, Jing-Ting; He, Kai-Yu; Li, Hong-Yu

    2007-05-01

    The complete sequences of 32 intergenic spacer regions (ISR) from Acidithiobacillus strains, including 29 field strains isolated from coal, copper, molybdenum mine wastes or sediment of different geoclimatic regions in China, reference strain ATCC19859 and the type strains of the two species were determined. These data, together with other sequences available in the GenBank database, were used to carry out the first detailed assessment of the inter- and intraspecific genomic variability of the ISR sequences and to infer phylogenetic relationships within the genus. The total length of the 16S-23S rRNA intergenic spacer regions of the Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans strains ranged from 451 to 490 bp, and from 434 to 456 bp, respectively. The degree of intrageneric ISR sequence similarity was higher than the degree of intergeneric similarity, and the overall similarity values of the ISRs varied from 60.49% to 84.71% between representatives of different species of the genus Acidithiobacillus. Sequences from the spacer of the A. thiooxidans and A. ferrooxidans strains ranged from 86.71% to 99.56% and 92.36% to 100% similarity, respectively. All Acidithiobacillus strains were separated into three phylogenetic major clusters and seven phylogenetic groups. ISR may be a potential target for the development of in situ hybridization probe aimed at accurately detecting acidithiobacilli in the various acidic environments.

  8. Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat.

    PubMed Central

    Parikh, V S; Conrad-Webb, H; Docherty, R; Butow, R A

    1989-01-01

    We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription. Images PMID:2473390

  9. Sequence variation in two mitochondrial DNA regions and internal transcribed spacer among isolates of the nematode Oesophagostomum asperum originating from goats in Hunan Province, China.

    PubMed

    Li, F; Hu, T; Duan, N C; Li, W Y; Teng, Q; Li, H; Liu, W; Liu, Y; Cheng, T Y

    2016-01-01

    The present study examined sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1), and internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) among Oesophagostomum asperum isolates from goats in Hunan Province, China. A portion of the cox1 (pcox1), nad1 (pnad1) genes and the ITS (ITS1+5.8S rDNA+ITS2) rDNA were amplified by polymerase chain reaction (PCR) separately from adult O. asperum individuals and the representative amplicons were subjected to sequencing from both directions. The lengths of pcox1, pnad1 and ITS rDNA were 366 bp, 681 bp and 785 bp, respectively. The A+T contents of gene sequences were 71.5-72% for pcox1, 73.7-74.2% for pnad1 and 58-58.8% for ITS rDNA. Intra-specific sequence variations within O. asperum were 0-1.6% for pcox1, 0-1.9% for pnad1 and 0-1.7% for ITS rDNA, while inter-specific sequence differences among members of the genus Oesophagostomum were significantly higher, being 11.1-12.5%, 13.3-17.7% and 8.5-18.6% for pcox1, pnad1 and ITS rDNA, respectively. Phylogenetic analyses using combined sequences of pcox1 and pnad1, with three different computational algorithms (Bayesian inference, maximum likelihood and maximum parsimony), revealed distinct groups with high statistical support. These findings demonstrated the existence of intra-specific variation in mtDNA and rDNA sequences among O. asperum isolates from goats in Hunan Province, China, and have implications for studying molecular epidemiology and population genetics of O. asperum.

  10. Internal transcribed spacer rRNA gene sequencing analysis of fungal diversity in Kansas City indoor environments

    PubMed Central

    Rittenour, William R.; Ciaccio, Christina E.; Barnes, Charles S.; Kashon, Michael L.; Lemons, Angela R.; Beezhold, Donald H.; Green, Brett J.

    2014-01-01

    Compared to traditional methods of fungal exposure assessment, molecular methods have provided new insight into the richness of fungal communities present in both indoor and outdoor environments. In this study, we describe the diversity of fungi in the homes of asthmatic children located in Kansas City. Fungal diversity was determined by sequencing the internal transcribed spacer (ITS) regions of ribosomal RNA derived from fungi collected in air and dust samples from 31 homes participating in the Kansas City Safe and Healthy Homes Program (KCSHHP). Sequencing results were then compared to data obtained using viable and non-viable fungal exposure assessment methods. ITS clone libraries were predominantly derived from the phylum Ascomycota in both air (68%) and dust (92%) samples and followed by the Basidiomycota and Zygomycota. The majority of Ascomycota clones belonged to four orders including the Pleosporales, Eurotiales, Capnodiales, and Dothideales. ITS sequencing revealed the presence of a number of rarely documented fungal species placed in the Pleosporales. Several species placed in the Basidiomycota were detected in ITS clone libraries but not by viable or non-viable methods. The prevalence of organizational taxonomic units (OTUs) was significantly higher in air than in dust samples (p < 0.0001); however, no differences between OTUs in air samples collected in the subjects’ room and basement were observed. These sequencing results demonstrate a much broader diversity of Ascomycota and Basidiomycota communities in KCSHHP indoor environments than previously estimated using traditional methods of assessment. PMID:24258337

  11. Roles of the tnaC-tnaA spacer region and Rho factor in regulating expression of the tryptophanase operon of Proteus vulgaris.

    PubMed Central

    Kamath, A V; Yanofsky, C

    1997-01-01

    To localize the DNA regions responsible for basal-level and induced expression of the tryptophanase (tna) operon of Proteus vulgaris, short deletions were introduced in the 115-bp spacer region separating tnaC, the leader peptide coding region, from tnaA. Deletions were incorporated into a tnaA'-'lacZ reporter construct containing the intact tna promoter-leader region. Expression was examined in Escherichia coli. Deletions that removed 28 to 30 bp from the region immediately following tnaC increased basal-level expression about threefold and allowed threefold induction by 1-methyltryptophan. A deletion removing 34 bp from the distal segment of the leader permitted basal and induced expression comparable to that of the parental construct. The mutant with the largest spacer deletion, 89 bp, exhibited a 30-fold increase in basal-level expression, and most importantly, inducer presence reduced operon expression by ca. 60%. Replacing the tnaC start codon or replacing or removing Trp codon 20 of tnaC of this deletion derivative eliminated inducer inhibition of expression. These findings suggest that the spacer region separating tnaC and tnaA is essential for Rho action. They also suggest that juxtaposition of the tnaC stop codon and the tnaA ribosome binding site in the 89-bp deletion derivative allows the ribosome that has completed translation of tnaC to inhibit translation initiation at the tnaA start codon when cells are exposed to inducer. These findings are consistent with results in the companion article that suggest that inducer allows the TnaC peptide to inhibit ribosome release at the tnaC stop codon. PMID:9045841

  12. Molecular genetics analysis for co-infection of Brugia malayi and Brugia pahangi in cat reservoirs based on internal transcribed spacer region 1.

    PubMed

    Areekit, Supatra; Khuchareontaworn, Sintawee; Kanjanavas, Pornpimon; Sriyapai, Thayat; Pakpitchareon, Arda; Khawsak, Paisarn; Chansiri, Kosum

    2009-01-01

    This study described the diagnosis of a mixed infection of Brugia malayi and Brugia pahangi in a single domestic cat using the internal transcribed spacer 1 (ITS1) region. Following polymerase chain reaction amplification of the ITS1 region, the 580 bp amplicon was cloned, and 29 white colonies were randomly selected for DNA sequencing and phylogenetic tree construction. A DNA parsimony tree generated two groups of Brugia spp with one group containing 6 clones corresponding to B. pahangi and the other 23 clones corresponding to B. malayi. This indicated that mixed infection of the two Brugia spp, B. pahangi and B. malayi, had occurred in a single host.

  13. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    NASA Astrophysics Data System (ADS)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  14. Detection of two Bartonella tamiae-like sequences in Amblyomma americanum (Acari: Ixodidae) using 16S-23S intergenic spacer region-specific primers.

    PubMed

    Billeter, Sarah A; Miller, Melissa K; Breitschwerdt, Edward B; Levy, Michael G

    2008-01-01

    Four hundred and sixty-six questing Amblyomma americanum (L.) (Acari: Ixodidae) from Carolina County, VA, and 98 questing A. americanum from Chatham County, NC, were screened by polymerase chain reaction (PCR) for the Bartonella 16S-23S intergenic spacer region. Two amplicons, approximately 270-280 bp, were detected in two ticks from Virginia. Based upon PCR and sequencing, an adult male and adult female tick harbored DNA sequences closely related to Bartonella tamiae (DQ395180). Bartonella DNA was not detected in A. americanum from North Carolina. Potential transmission of Bartonella spp. by A. americanum should be the focus of future experimental studies.

  15. Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae) of the Andean region

    PubMed Central

    Sutton, Bruce D.; Steck, Gary J.; Norrbom, Allen L.; Rodriguez, Erick J.; Srivastava, Pratibha; Alvarado, Norma Nolazco; Colque, Fredy; Landa, Erick Yábar; Sánchez, Juan José Lagrava; Quisberth, Elizabeth; Peñaranda, Emilio Arévalo; Clavijo, P. A. Rodriguez; Alvarez-Baca, Jeniffer K.; Zapata, Tito Guevara; Ponce, Patricio

    2015-01-01

    Abstract The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The ITS1 regions of additional specimens (17 collections) from Central America (México, Guatemala, Costa Rica, and Panamá), Brazil, Caribbean Colombia, and coastal Venezuela were sequenced and together with published sequences (Paraguay) provided context for interpretation. A total of six ITS1 sequence variants were recognized in the Andean region comprising four groups. Type I predominates in the southernmost range of Anastrepha fraterculus. Type II predominates in its northernmost range. In the central and northern Andes, the geographic distributions overlap and interdigitate with a strong elevational effect. A discussion of relationships between observed ITS1 types and morphometric types is included. PMID:26798259

  16. Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing.

    PubMed

    Volokhov, Dmitriy V; George, Joseph; Liu, Sue X; Ikonomi, Pranvera; Anderson, Christine; Chizhikov, Vladimir

    2006-08-01

    We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction-microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.

  17. Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae) of the Andean region.

    PubMed

    Sutton, Bruce D; Steck, Gary J; Norrbom, Allen L; Rodriguez, Erick J; Srivastava, Pratibha; Alvarado, Norma Nolazco; Colque, Fredy; Landa, Erick Yábar; Sánchez, Juan José Lagrava; Quisberth, Elizabeth; Peñaranda, Emilio Arévalo; Clavijo, P A Rodriguez; Alvarez-Baca, Jeniffer K; Zapata, Tito Guevara; Ponce, Patricio

    2015-01-01

    The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The ITS1 regions of additional specimens (17 collections) from Central America (México, Guatemala, Costa Rica, and Panamá), Brazil, Caribbean Colombia, and coastal Venezuela were sequenced and together with published sequences (Paraguay) provided context for interpretation. A total of six ITS1 sequence variants were recognized in the Andean region comprising four groups. Type I predominates in the southernmost range of Anastrepha fraterculus. Type II predominates in its northernmost range. In the central and northern Andes, the geographic distributions overlap and interdigitate with a strong elevational effect. A discussion of relationships between observed ITS1 types and morphometric types is included.

  18. Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    Takahashi, Mai; Yukphan, Pattaraporn; Yamada, Yuzo; Suzuki, Ken-ichiro; Sakane, Takeshi; Nakagawa, Yasuyoshi

    2006-06-01

    Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.

  19. Effect of the nature of the spacer on gene transfer efficacies of novel thiocholesterol derived gemini lipids in different cell lines: a structure-activity investigation.

    PubMed

    Bajaj, Avinash; Kondaiah, Paturu; Bhattacharya, Santanu

    2008-04-24

    A structure-activity investigation was undertaken to see the effect of the nature of the spacer on the gene transfection efficacies of thiocholesterol-derived cationic gemini lipids possessing disulfide linkage between the cationic headgroup and the thiocholesterol moiety. Three gemini cationic lipids possessing hydrophobic flexible (-(CH 2) 5-; 1), hydrophobic rigid (-C 6H 4-; 2), and hydrophilic flexible (-CH 2-CH 2-O-CH 2-CH 2-; 3) spacer segments were synthesized. In HeLa cells, lipid formulations 1 and 2 were found to be more effective as compared to lipid 3 formulation. In HT1080 cell line, the order of transfectability was 3 > 1 > 2. Transfection studies in HeLa and HT1080 cell line also showed 40-50% transfection efficacy in the presence of 10% serum conditions. These formulations were also able to transfect gene across difficult cells like HaCaT. Cytotoxic studies showed the nontoxic nature of these lipid-DNA complexes at different N/P ratios used for transfection studies.

  20. Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers.

    PubMed

    Suezawa, Yasuhiko; Suzuki, Motofumi; Mori, Haruhiko

    2008-09-01

    We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.

  1. [Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus].

    PubMed

    Slugina, M A; Torres Minho, K; Filiushin, M A

    2014-01-01

    Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5'-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.

  2. Amplification of the 16S-23S rDNA spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum.

    PubMed

    Sasaki, Y; Yamamoto, K; Amimoto, K; Kojima, A; Ogikubo, Y; Norimatsu, M; Ogata, H; Tamura, Y

    2001-12-01

    Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens.

  3. Use of specific primers based on the 16S-23S internal transcribed spacer (ITS) region for the screening Bifidobacterium adolescentis in yogurt products and human stool samples.

    PubMed

    Tsai, Cheng-Chih; Lai, Chieh-Hsien; Yu, Bi; Tsen, Hau-Yang

    2008-10-01

    Effective methods for the identification and enumeration of lactic acid producing bacteria (LAB) cells are important for the quality control and assurance of probiotic products. In this study, we designed a polymerase chain reaction (PCR) primer set from the sequence in 16S-23S internal transcribed spacer (ITS) region and used it for the specific detection of Bifidobacterium adolescentis, one of the Bifidobacterium species used in probiotics. Specificity of the PCR primers, i.e., bits-1/bits-2, was assured by assay strains of B. adolescentis, other Bifidobacterium species, and strains of non-Bifidobacterium spp. Coupled with the use of a known primer set specific for Bifidobacterium species, Bifidobacterium strains and B. adolescentis could be identified from LAB strains in fermented dairy products and human fecal samples.

  4. Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron.

    PubMed

    Elbadri, Gamal A A; De Ley, Paul; Waeyenberge, Lieven; Vierstraete, Andy; Moens, Maurice; Vanfleteren, Jacques

    2002-02-01

    Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and 'Radopholus citrophilus'.

  5. Sequence variation of the ribosomal DNA second internal transcribed spacer region in two spatially-distinct populations of Amblyomma americanum (L.) (Acari: Ixodidae).

    PubMed

    Reichard, M V; Kocan, A A; Van Den Bussche, R A; Barker, R W; Wyckoff, J H; Ewing, S A

    2005-04-01

    Sequence analysis of the ribosomal DNA second internal transcribed spacer (ITS 2) region in 2 spatially distinct populations of Amblyomma americanum (L.) revealed intraspecific variation. Nucleotide sequences from multiple DNA extractions and several polymerase chain reaction amplifications of eggs from mixed-parentage samples from both populations of ticks revealed that 12 of 1,145 (1.0%) sites varied. Three of the 12 sites of variation were distinct between the 2 A. americanum populations, which corresponded to a rate of 0.26%. Phylogenetic analysis based on ITS 2 sequences provided strong support (i.e., bootstrap value of 80%) that wild A. americanum clustered into a distinguishable group separate from those derived from colony ticks.

  6. Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA

    PubMed Central

    Gupta, Aditya K.; Boekhout, Teun; Theelen, Bart; Summerbell, Richard; Batra, Roma

    2004-01-01

    Malassezia yeasts are associated with several dermatological disorders. The conventional identification of Malassezia species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular techniques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal transcribed spacer, and sequencing of the D1 and D2 domains of the large-subunit ribosomal DNA region, was applied for the identification of Malassezia species. All species could be correctly identified by means of these methods. The results of AFLP analysis and sequencing were in complete agreement with each other. However, some discrepancies were noted when the molecular methods were compared with the phenotypic method of identification. Specific genotypes were distinguished within a collection of Malassezia furfur isolates from Canadian sources. AFLP analysis revealed significant geographical differences between the North American and European M. furfur strains. PMID:15365020

  7. Conservation in the first internal transcribed spacer region (ITS1) in Hematodinium species infecting crustacean hosts found in the UK and Newfoundland.

    PubMed

    Small, H J; Shields, J D; Moss, J A; Reece, K S

    2007-05-09

    Parasitic dinoflagellates in the genus Hematodinium infect a number of decapod crustaceans in waters off the UK, including the Norway lobster Nephrops norvegicus and the edible crab Cancer pagurus. This study investigated sequence variability in the first internal transcribed spacer (ITS1) region of the ribosomal RNA complex of Hematodinium spp. infecting N. norvegicus, C. pagurus, and Pagurus bernhardus from 4 locations in the UK and from the Hematodinium sp. infecting Chionoecetes opilio from the province of Newfoundland and Labrador, Canada. Phylogenetic analysis of the Hematodinium ITS1 sequences from N. norvegicus, C. pagurus, P. bernhardus and C. opilio suggest that these crustaceans are infected with the same species of Hematodinium. Length variability of the ITS1 region was observed (324 to 345 bp) and attributed to 4 variable microsatellite regions (CATG)n' (GCC)nTCCGC(TG)n' (TA)n' and (GAA)n(GGA)n within the sequenced ITS1 fragment. The observed variation may be due to co-infection of the host crustacean with several different strains of Hematodinium or differences among copies of ITS1 region within the genome of a single parasite cell. The Hematodinium ITS1 sequence from N. norvegicus, C. pagurus, P. bernhardus and C. opilio isolates was sufficiently conserved in primer binding regions targeted by previous molecular diagnostic assays; therefore, we suggest that this assay could be used to screen for Hematodinium infections in these crustacean hosts.

  8. Extensive 5.8S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions.

    PubMed

    Harpke, Doerte; Peterson, Angela

    2008-05-01

    The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems.

  9. Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer (ITS) region of peritrich ciliates in environmental samples

    NASA Astrophysics Data System (ADS)

    Su, Lei; Zhang, Qianqian; Gong, Jun

    2017-07-01

    Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifications of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.

  10. Host selectivity and genetic variation of Discula umbrinella isolates from two oak species: analyses of intergenic spacer region sequences of ribosomal DNA.

    PubMed

    Cohen, Susan D

    2006-10-01

    Discula umbrinella, a fungal endophyte of oak species, colonizes and reproduces on leaves of Quercus alba and Q. rubra in forest ecosystems. Twenty-nine isolates collected from leaves of both oak species (16 from Q. alba and 13 from Q. rubra) were assayed for oak species preference and genetic variation based on primer-specific polymerase chain reactions for the intergenic spacer region (IGS) of ribosomal DNA. DNA sequencing of the polymerase chain reaction products revealed a 10-bp insertion (237-247 bp) at the 3' end of the IGS region present in nine isolates and absent in 20 of the isolates. Phylogenetic analysis of the IGS region using the neighbor-joining method identified IGS groups (groups I-V) based on single nucleotide sequence differences. Host selectivity and geographic origin of isolates were correlated in some instances with the IGS groups. Isolates within each IGS group were further analyzed for nucleotide polymorphisms to confirm genotype identity and genotype diversity. Ten different genotypes (Va-Vj) were identified among the isolates analyzed. Genotype diversity was greatest in IGS groups I, IV, and V. Seventy percent of the genotypes (Vc, Vd, Ve, Vf, Vg,Vi, and Vj) contained isolates with single tree species preferences.

  11. Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains.

    PubMed

    Haga, S; Hirano, Y; Murayama, O; Millar, B C; Moore, J E; Matsuda, M

    2003-01-01

    To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.

  12. Development of internal transcribed spacer regions amplification restriction fragment length polymorphism method and its application in monitoring the population of Zygosaccharomyces rouxii M2 in miso fermentation.

    PubMed

    Sujaya, I Nengah; Tamura, Yoshifumi; Tanaka, Takayuki; Yamaki, Tazusa; Ikeda, Takayuki; Kikushima, Nao; Yata, Hiroshi; Yokota, Atsushi; Asano, Kozo; Tomita, Fusao

    2003-01-01

    Recently, the use of the dry yeast of Zygosaccharomyces rouxii M2 for miso (soybean paste) fermentation has been established. A molecular monitoring method was developed and validated in this study to analyze the population of Z. rouxii M2 during the fermentation. The method was based on the restriction patterns of internal transcribed spacer (ITS) regions of the rDNA using HaeIII and HhaI. Among the homologous ITS regions of Z. rouxii strains, Z. rouxii M2 produced diagnostic bands by which it can be differentiated from the other strains used. The specific restriction bands were due to the difference in nucleotide sequence of two different copies of ITS of Z. rouxii M2. Both ITS copies showed 94% sequence similarity but a 13-bp nucleotide substitution and a 19-bp deletion were found in the ITS1 region. Phylogenetic trees were constructed based on ITS and 18S rDNA sequences and it was found that the ITS sequences provide better resolution for the classification of Z. rouxii M2. Since Z. rouxii M2 is a promising strain for use in miso fermentation as a dry starter, the method developed is significant in terms of industrial application in monitoring the growth of Z. rouxii M2 in miso fermentation.

  13. Intraspecific variation and population structure of the German cockroach, Blattella germanica, revealed with RFLP analysis of the non-transcribed spacer region of ribosomal DNA.

    PubMed

    Mukha, D V; Kagramanova, A S; Lazebnaya, I V; Lazebnyi, O E; Vargo, E L; Schal, C

    2007-06-01

    Little information is available on genetic variation within and between populations of pest cockroaches. In this study, intraspecific HindIII polymorphism was investigated in the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera, Blattaria: Blattellidae), using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of ribosomal DNA (rDNA). Individual male insects were collected from infestations at three different pig farms. Each population was characterized by HindIII restriction fragment frequencies and haplotype (a particular X-chromosome pattern) frequencies. The inheritance of the X-chromosome HindIII rDNA patterns over 12 generations (3 years) follows Mendelian patterns, and the stability of this polymorphic marker indicates infrequent genetic recombination of variable sites. Although pairwise genetic distance measures were uncorrelated with geographical distance, the pattern of genetic differentiation of the three cockroach populations suggests that human-mediated transport of cockroaches is an important force in shaping the population genetic structure of cockroach infestations, at least at the regional scale of 10-100 km. Sequence variation in the ribosomal NTS is a useful marker, and RFLP of rDNA is a simple, robust and reproducible technique for differentiating recently diverged cockroach populations.

  14. Molecular characterization of the Indian poultry nodular tapeworm, Raillietina echinobothrida (Cestoda: Cyclophyllidea: Davaineidae) based on rDNA internal transcribed spacer 2 region.

    PubMed

    Ramnath; Jyrwa, D B; Dutta, A K; Das, B; Tandon, V

    2014-03-01

    The nodular tapeworm, Raillietina echinobothrida is a well studied avian gastrointestinal parasite of family Davaineidae (Cestoda: Cyclophyllidea). It is reported to be the largest in size and second most prevalent species infecting chicken in north-east India. In the present study, morphometrical methods coupled with the molecular analysis of the second internal transcribed spacer (ITS2) region of ribosomal DNA were employed for precise identification of the parasite. The annotated ITS2 region was found to be 446 bp long and further utilized to elucidate the phylogenetic relationships and its species-interrelationships at the molecular level. In phylogenetic analysis similar topology was observed among the trees obtained by distance-based neighbor-joining as well as character-based maximum parsimony tree building methods. The query sequence R. echinobothrida is well aligned and placed within the Davaineidae group, with all Raillietina species well separated from the other cyclophyllidean (taeniid and hymenolepid) cestodes, while Diphyllobothrium latum (Pseudophyllidea: Diphyllobothriidae) was rooted as an out-group. Sequence similarities indeed confirmed our hypothesis that Raillietina spp. are neighboring the position with other studied species of order Cyclophyllidea against the out-group order Pseudophyllidea. The present study strengthens the potential of ITS2 as a reliable marker for phylogenetic reconstructions.

  15. Intragenomic heterogeneity of a ribosomal DNA spacer (ITS2) varies regionally in the neotropical malaria vector Anopheles nuneztovari (Diptera:Culicidae).

    PubMed

    Onyabe, D Y; Conn, J E

    1999-11-01

    We investigated intragenomic heterogeneity of ITS2 within twenty individual Anopheles nuneztovari (subgenus Nyssorhynchus) from five geographical localities in the neotropics (three from Brazil and one each from Colombia and Venezuela) by cloning and sequencing PCR-amplified copies of this spacer. Intragenomic heterogeneity was observed in thirteen of twenty mosquitoes of both sexes from all localities. As estimated by uncorrected P, however, mean sequence divergence was greater in mosquitoes from Brazil (PR = 0.0100, BL = 0.0196, AB = 0.0182) than in those from Venezuela (SO = 0.0026) or Colombia (SI = 0.0078). Sequence divergence per genome was significantly higher in mosquitoes from Brazil than in those from SO and SI. In fact, divergence among ITS2 variants within single mosquitoes from the Brazilian localities was often as great as that between localities. Similarly, the number of variants per mosquito was significantly greater in Brazil (maximum of six variants) than in both SO and SI (maximum of two variants). These results indicate that homogenization of ITS2 has proceeded to a greater extent in SO and SI than in the localities in Brazil. The differences in intragenomic heterogeneity between both SO and SI versus the Brazilian localities probably reflect either differences in population-level processes such as gene flow and genetic drift, or the fact that these localities may represent two or more cryptic species, as suggested by other studies.

  16. Molecular identification of the Indian liver fluke, Fasciola (Trematoda: Fasciolidae) based on the ribosomal internal transcribed spacer regions.

    PubMed

    Prasad, P K; Tandon, V; Biswal, D K; Goswami, L M; Chatterjee, A

    2008-11-01

    The species of liver flukes of the genus Fasciola (Platyhelminthes: Digenea: Fasciolidae) are obligate parasitic trematodes residing in the large biliary ducts of herbivorous mammals. While Fasciola hepatica has a cosmopolitan distribution, the other major species, i.e., Fasciola gigantica is reportedly prevalent in the tropical and subtropical regions of Africa and Asia. To determine the phylogenic location of Fasciola sp. of Assam (India) origin based on rDNA molecular data, ribosomal ITS regions were sequenced and compared with other species of trematodes in the family Fasciolidae. NCBI databases were used for sequence homology analysis using BLAST and ClustalW programs. The phylogenetic trees constructed based upon the ITS (1 and 2) sequences revealed a close relationship with isolates of F. gigantica from China, Indonesia, Japan, Egypt, and Zambia, the isolate from China with significant bootstrap values being the closest. Using the novel approach of molecular morphometrics that is based on ITS2 secondary structure homologies, phylogenetic relationships of the various isolates of fasciolid species have also been discussed. While comparing ITS1, the sequence of another Indian isolate designated as F. gigantica (accession no. EF198867) showed almost absolute match with F. hepatica. Hence, this particular isolate should be identified as F. hepatica and not F. gigantica.

  17. Rapid and direct detection of clostridium chauvoei by PCR of the 16S-23S rDNA spacer region and partial 23S rDNA sequences.

    PubMed

    Sasaki, Y; Yamamoto, K; Kojima, A; Tetsuka, Y; Norimatsu, M; Tamura, Y

    2000-12-01

    Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.

  18. Molecular variation analysis of Aspergillus flavus using polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer rDNA region

    PubMed Central

    Zarrin, Majid; Erfaninejad, Maryam

    2016-01-01

    Aspergillus flavus is the second most common disease-causing species of Aspergillus in humans. The fungus is frequently associated with life-threatening infections in immunocompromised hosts. The primary aim of the present study was to analyze the genetic variability among different isolates of A. flavus using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A total of 62 A. flavus isolates were tested in the study. Molecular variability was searched for by analysis of the PCR amplification of the internal transcribed spacer (ITS) regions of ribosomal DNA using restriction enzymes. PCR using primers for ITS1 and ITS4 resulted in a product of ~600 bp. Amplicons were subjected to digestion with restriction endonucleases EcoRI, HaeIII and TaqI. Digestion of the PCR products using these restriction enzymes produced different patterns of fragments among the isolates, with different sizes and numbers of fragments, revealing genetic variability. In conclusion, ITS-RFLP is a useful molecular tool in screening for nucleotide polymorphisms among A. flavus isolates. PMID:27588085

  19. Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

    PubMed

    Tavares, Eliandro Reis; Azevedo, Caroline Souza; Panagio, Luciano Aparecido; Pelisson, Marsileni; Pinge-Filho, Phileno; Venancio, Emerson José; Barros, Tânia Fraga; Yamada-Ogatta, Sueli Fumie; Yamauchi, Lucy Megumi

    2016-01-01

    In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

  20. Analysis of the rDNA internal transcribed spacer region of the Fusarium species by polymerase chain reaction-restriction fragment length polymorphism

    PubMed Central

    ZARRIN, MAJID; GANJ, FARZANEH; FARAMARZI, SAMA

    2016-01-01

    The Fusarium species are a widely spread phytopathogen identified in an extensive variety of hosts. The Fusarium genus is one of the most heterogeneous fungi and is difficult to classify. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is a useful method in detection of DNA polymorphism in objective sequences. The aim of the present study was to identify the phylogenetic associations and usefulness of the internal transcribed spacer (ITS) region as a genetic marker within the most clinically important strain of the Fusarium species. A total of 50 strains of Fusarium spp. were used in the study, including environmental, clinical and reference isolates. The primers ITS1 and ITS4 were used in the study. Two restriction enzymes, HaeIII and SmaI, were assessed for the digestion of PCR products. A PCR product of ~550-base pairs was generated for each Fusarium species. The digested products with HaeIII and SmaI demonstrated that the bands generated for the medically significant Fusarium species, including F. solani, F. oxysporum, F. verticillidea, F. proliferatum and F. fujikuri, have different restriction enzyme patterns. In conclusion, it appears that the PCR-RFLP method used in the present study produces a sufficient restriction profile for differentiation of the most medically significant Fusarium species. PMID:27073635

  1. Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS).

    PubMed

    Wrent, Petra; Rivas, Eva-María; Peinado, José M; de Silóniz, María-Isabel

    2010-08-15

    Unlike previously reported methods that need a combination of several typing techniques, we have developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage species. Strains belonging to other species have also been included for comparison. We have demonstrated that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z. bailii. We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of Zygosaccharomyces in a separate cluster.

  2. DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions.

    PubMed

    Dingman, Douglas W

    2009-01-01

    Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.

  3. From Genus to Phylum: Large-Subunit and Internal Transcribed Spacer rRNA Operon Regions Show Similar Classification Accuracies Influenced by Database Composition

    PubMed Central

    Liu, Kuan-Liang; Kuske, Cheryl R.

    2014-01-01

    We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5′ section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets. PMID:24242255

  4. Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

    PubMed Central

    Toro-Ascuy, Daniela; Tambley, Carolina; Beltran, Carolina; Mascayano, Carolina; Sandoval, Nicolas; Olivares, Eduardo; Medina, Rafael A.; Spencer, Eugenio

    2014-01-01

    Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV901_09 and rISAVrS6-NotI-HPR containing a NotI restriction site and rISAVS6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 105 PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry. PMID:25480750

  5. From genus to phylum: large-subunit and internal transcribed spacer rRNA operon regions show similar classification accuracies influenced by database composition.

    PubMed

    Porras-Alfaro, Andrea; Liu, Kuan-Liang; Kuske, Cheryl R; Xie, Gary

    2014-02-01

    We compared the classification accuracy of two sections of the fungal internal transcribed spacer (ITS) region, individually and combined, and the 5' section (about 600 bp) of the large-subunit rRNA (LSU), using a naive Bayesian classifier and BLASTN. A hand-curated ITS-LSU training set of 1,091 sequences and a larger training set of 8,967 ITS region sequences were used. Of the factors evaluated, database composition and quality had the largest effect on classification accuracy, followed by fragment size and use of a bootstrap cutoff to improve classification confidence. The naive Bayesian classifier and BLASTN gave similar results at higher taxonomic levels, but the classifier was faster and more accurate at the genus level when a bootstrap cutoff was used. All of the ITS and LSU sections performed well (>97.7% accuracy) at higher taxonomic ranks from kingdom to family, and differences between them were small at the genus level (within 0.66 to 1.23%). When full-length sequence sections were used, the LSU outperformed the ITS1 and ITS2 fragments at the genus level, but the ITS1 and ITS2 showed higher accuracy when smaller fragment sizes of the same length and a 50% bootstrap cutoff were used. In a comparison using the larger ITS training set, ITS1 and ITS2 had very similar accuracy classification for fragments between 100 and 200 bp. Collectively, the results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets.

  6. Loop-Mediated Isothermal Amplification for Detection of the 5.8S Ribosomal Ribonucleic Acid Internal Transcribed Spacer 2 Gene Found in Trypanosoma brucei gambiense.

    PubMed

    Nikolskaia, Olga V; Thekisoe, Oriel M M; Dumler, J Stephen; Grab, Dennis J

    2017-02-08

    The loop-mediated isothermal amplification (LAMP) assay with its advantages of cost effectiveness, rapidity, and simplicity, has evolved as a sensitive and specific method for the detection of African trypanosomes. Highly sensitive LAMP reactions specific for Trypanosoma brucei rhodesiense or that recognize but do not discriminate between Trypanosoma brucei brucei, T. b. rhodesiense, Trypanosoma brucei gambiense, and Trypanosoma evansi have been developed. A sensitive LAMP assay targeting the T. b. gambiense 5.8S ribosomal RNA internal transcribed spacer 2 (5.8S-ITS2) gene is also available but this assay does not target binding sites that span the CCCA (C3A) (557-560 bps) insertion site that further differentiates T. b. gambiense from T. b. brucei Here we describe 5.8S-ITS2-targeted LAMP assay that fit these criteria. The LAMP primer sets containing the T. b. gambiense-specific C3A tetranucleotide at the start of the outer forward primer sequences showed high specificity and sensitivity down to at least 0.1 fg T. b. gambiense genomic DNA. © The American Society of Tropical Medicine and Hygiene.

  7. A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA▿

    PubMed Central

    Pecson, Brian M.; Barrios, José Antonio; Johnson, David R.; Nelson, Kara L.

    2006-01-01

    Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications. PMID:17056687

  8. Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

    PubMed Central

    Chen, Sharon C.-A.; Wang, He; Zhang, Li; Fan, Xin; Xu, Zhi-Peng; Cheng, Jing-Wei; Kong, Fanrong; Zhao, Yu-Pei; Xu, Ying-Chun

    2016-01-01

    Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species. PMID:27105313

  9. Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

    PubMed Central

    Roth, Andreas; Reischl, Udo; Streubel, Anna; Naumann, Ludmila; Kroppenstedt, Reiner M.; Habicht, Marion; Fischer, Marga; Mauch, Harald

    2000-01-01

    A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S r

  10. Touchdown Enzyme Time Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci Using the 16S and 16S-23S Spacer rRNA Genes

    PubMed Central

    Madico, Guillermo; Quinn, Thomas C.; Boman, Jens; Gaydos, Charlotte A.

    2000-01-01

    Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (κ, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples. PMID:10699002

  11. CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication.

    PubMed

    Yang, Chi-Dung; Chen, Yen-Hua; Huang, Hsi-Yuan; Huang, Hsien-Da; Tseng, Ching-Ping

    2014-06-01

    The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.

  12. Novel genetic diversity within Anopheles punctimacula s.l.: phylogenetic discrepancy between the Barcode cytochrome c oxidase I (COI) gene and the rDNA second internal transcribed spacer (ITS2).

    PubMed

    Loaiza, Jose R; Scott, Marilyn E; Bermingham, Eldredge; Sanjur, Oris I; Rovira, Jose R; Dutari, Larissa C; Linton, Yvonne-Marie; Bickersmith, Sara; Conn, Jan E

    2013-10-01

    Anopheles punctimacula s.l. is a regional malaria vector in parts of Central America, but its role in transmission is controversial due to its unresolved taxonomic status. Two cryptic species, An. malefactor and An. calderoni, have been previously confused with this taxon, and evidence for further genetic differentiation has been proposed. In the present study we collected and morphologically identified adult female mosquitoes of An. punctimacula s.l. from 10 localities across Panama and one in Costa Rica. DNA sequences from three molecular regions, the three prime end of the mitochondrial cytochrome c oxidase I gene (3' COI), the Barcode region in the five prime end of the COI (5' COI), and the rDNA second internal transcribed spacer (ITS2) were used to test the hypothesis of new molecular lineages within An. punctimacula s.l. Phylogenetic analyses using the 3' COI depicted six highly supported molecular lineages (A-F), none of which was An. malefactor. In contrast, phylogenetic inference with the 5' COI demonstrated paraphyly. Tree topologies based on the combined COI regions and ITS2 sequence data supported the same six lineages as the 3' COI alone. As a whole this evidence suggests that An. punctimacula s.l. comprises two geographically isolated lineages, but it is not clear whether these are true species. The phylogenetic structure of the An. punctimacula cluster as well as that of other unknown lineages (C type I vs C type II; D vs E) appears to be driven by geographic partition, because members of these assemblages did not overlap spatially. We report An. malefactor for the first time in Costa Rica, but our data do not support the presence of An. calderoni in Panama. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae) of the Andean region

    USDA-ARS?s Scientific Manuscript database

    The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The IT...

  14. Co-infection of Haemonchus contortus and Trichostrongylus spp. among livestock in Malaysia as revealed by amplification and sequencing of the internal transcribed spacer II DNA region

    PubMed Central

    2014-01-01

    Background Haemonchus contortus and Trichostrongylus spp. are reported to be the most prevalent and highly pathogenic parasites in livestock, particularly in small ruminants. However, the routine conventional tool used in Malaysia could not differentiate the species accurately and therefore limiting the understanding of the co-infections between these two genera among livestock in Malaysia. This study is the first attempt to identify the strongylids of veterinary importance in Malaysia (i.e., H. contortus and Trichostrongylus spp.) by amplification and sequencing of the Internal Transcribed Spacer II DNA region. Results Overall, 118 (cattle: 11 of 98 or 11.2%; deer: 4 of 70 or 5.7%; goats: 99 of 157 or 63.1%; swine: 4 of 91 or 4.4%) out of the 416 collected fecal samples were microscopy positive with strongylid infection. The PCR and sequencing results demonstrated that 93 samples (1 or 25.0% of deer; 92 or 92.9% of goats) contained H. contortus. In addition, Trichostrongylus colubriformis was observed in 75 (75.8% of 99) of strongylid infected goats and Trichostrongylus axei in 4 (4.0%) of 99 goats and 2 (50.0%) of 4 deer. Based on the molecular results, co-infection of H. contortus and Trichostrongylus spp. (H. contortus + T. colubriformis denoted as HTC; H. contortus + T. axei denoted as HTA) were only found in goats. Specifically, HTC co-infections have higher rate (71 or 45.2% of 157) compared to HTA co-infections (3 or 1.9% of 157). Conclusions The present study is the first molecular identification of strongylid species among livestock in Malaysia which is essential towards a better knowledge of the epidemiology of gastro-intestinal parasitic infection among livestock in the country. Furthermore, a more comprehensive or nationwide molecular-based study on gastro-intestinal parasites in livestock should be carried out in the future, given that molecular tools could assist in improving diagnosis of veterinary parasitology in Malaysia due to its high

  15. The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers.

    PubMed

    Luo, Michelle L; Jackson, Ryan N; Denny, Steven R; Tokmina-Lukaszewska, Monika; Maksimchuk, Kenneth R; Lin, Wayne; Bothner, Brian; Wiedenheft, Blake; Beisel, Chase L

    2016-09-06

    Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.

  16. The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

    PubMed Central

    Luo, Michelle L.; Jackson, Ryan N.; Denny, Steven R.; Tokmina-Lukaszewska, Monika; Maksimchuk, Kenneth R.; Lin, Wayne; Bothner, Brian; Wiedenheft, Blake; Beisel, Chase L.

    2016-01-01

    Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity. PMID:27174938

  17. The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

    DOE PAGES

    Luo, Michelle L.; Jackson, Ryan N.; Denny, Steven R.; ...

    2016-05-12

    Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of thismore » complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Lastly, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.« less

  18. Promoter region of mouse Tcrg genes

    SciTech Connect

    Ishimi, Y.; Huang, Y.Y.; Ohta, S.

    1996-06-01

    The mouse T-cell receptor (Tcr){gamma} chain is characterized by a specific expression of V gene segments in the thymus corresponding to consecutive developmental stages; i.e., the Vg5 in fetal, Vg6 in neonatal, and Vg4 and Vg7 in adult. The order of the Vg gene usage correlates with the localization of the Vg gene segment on the chromosome; i.e., the Vg5 gene, being most proximal to the Jg1, is used first, followed by the Vg segments away from the Jg1 in a sequential manner. Since they all rearrange to the same Jg1 gene segment, the sequences in the coding region and/or in the 5{prime} upstream region are responsible for the stage-specific transcription. Also, Goldman and co-workers reported the germline transcription of Vg genes preceding their rearrangement. Therefore, the stage-specific transcription may be involved in the regulation of the stage-specific rearrangement; we sequenced and analyzed the 5{prime} flanking regions of the Vg5, Vg6, Vg4, and Vg7 genes to study the transcriptional relation. 18 refs., 2 figs., 1 tab.

  19. Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

    Treesearch

    Jill E. Petrisko; Christopher A. Pearl; David S. Pilliod; Peter P. Sheridan; Charles F. Williams; Charles R. Peterson; R. Bruce Bury

    2008-01-01

    We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We...

  20. Gene Regions Responding to Skeletal Muscle Atrophy

    NASA Technical Reports Server (NTRS)

    Booth, Frank W.

    1997-01-01

    Our stated specific aims for this project were: 1) Identify the region(s) of the mouse IIb myosin heavy chain (MHC) promoter necessary for in vivo expression in mouse fast-twitch muscle, and 2) Identify the region(s) of the mouse IIb MHC promoter responsive to immobilization in mouse slow-twitch muscle in vivo. We sought to address these specific aims by introducing various MHC IIb promoter/reporter gene constructs directly into the tibialis anterior and gastrocnemius muscles of living mice. Although the method of somatic gene transfer into skeletal muscle by direct injection has been successfully used in our laboratory to study the regulation of the skeletal alpha actin gene in chicken skeletal muscle, we had many difficulties utilizing this procedure in the mouse. Because of the small size of the mouse soleus and the difficulty in obtaining consistent results, we elected not to study this muscle as first proposed. Rather, our MHC IIb promoter deletion experiments were performed in the gastrocnemius. Further, we decided to use hindlimb unloading via tail suspension to induce an upregulation of the MHC IIb gene, rather than immobilization of the hindlimbs via plaster casts. This change was made because tail suspension more closely mimics spaceflight, and this procedure in our lab results in a smaller loss of overall body mass than the mouse hindlimb immobilization procedure. This suggests that the stress level during tail suspension is less than during immobilization. This research has provided an important beginning point towards understanding the molecular regulation of the MHC lIb gene in response to unweighting of skeletal muscle Future work will focus on the regulation of MHC IIb mRNA stability in response to altered loading of skeletal muscle

  1. Effect of spacer layers on current-voltage characteristics of resonant-tunneling diode

    SciTech Connect

    Remnev, M. A. Kateev, I. Yu.; Elesin, V. F.

    2010-08-15

    Using the numerical solution to the Schroedinger equation, current-voltage characteristics of the resonant-tunneling diode with spacer layers were obtained. The dependences of the peak current of the resonant-tunneling diode on the emitter spacer width were plotted. It was shown that the peak current depends periodically on the emitter spacer width. The constructed electron density diagrams showed that the increase in the peak current is associated with the resonant level in the emitter spacer region.

  2. Bioinformatics analyses of Shigella CRISPR structure and spacer classification.

    PubMed

    Wang, Pengfei; Zhang, Bing; Duan, Guangcai; Wang, Yingfang; Hong, Lijuan; Wang, Linlin; Guo, Xiangjiao; Xi, Yuanlin; Yang, Haiyan

    2016-03-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of a variety of archaea and bacteria and indicative of the bacterial ecological adaptation, conferring acquired immunity against invading foreign nucleic acids. Shigella is an important pathogen for anthroponosis. This study aimed to analyze the features of Shigella CRISPR structure and classify the spacers through bioinformatics approach. Among 107 Shigella, 434 CRISPR structure loci were identified with two to seven loci in different strains. CRISPR-Q1, CRISPR-Q4 and CRISPR-Q5 were widely distributed in Shigella strains. Comparison of the first and last repeats of CRISPR1, CRISPR2 and CRISPR3 revealed several base variants and different stem-loop structures. A total of 259 cas genes were found among these 107 Shigella strains. The cas gene deletions were discovered in 88 strains. However, there is one strain that does not contain cas gene. Intact clusters of cas genes were found in 19 strains. From comprehensive analysis of sequence signature and BLAST and CRISPRTarget score, the 708 spacers were classified into three subtypes: Type I, Type II and Type III. Of them, Type I spacer referred to those linked with one gene segment, Type II spacer linked with two or more different gene segments, and Type III spacer undefined. This study examined the diversity of CRISPR/cas system in Shigella strains, demonstrated the main features of CRISPR structure and spacer classification, which provided critical information for elucidation of the mechanisms of spacer formation and exploration of the role the spacers play in the function of the CRISPR/cas system.

  3. Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA

    PubMed Central

    2014-01-01

    Background Molecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene. Methods Mosquitoes were collected, identified, dissected to check infection status, and DNA extraction was performed for PCR with primers targeting the ITS2 rDNA region. Sequencing was done and phylogenetic tree was constructed to study the evolutionary relationship among Anopheles mosquitoes within Peninsular Malaysia, as well as across the Asian region. Results A total of 133 Anopheles mosquitoes consisting of six different species were collected from eight different locations across Peninsular Malaysia. Of these, 65 ITS2 rDNA sequences were obtained. The ITS2 rDNA amplicons of the studied species were of different sizes. One collected species, Anopheles sinensis, shows two distinct pools of population in Peninsular Malaysia, suggesting evolvement of geographic race or allopatric speciation. Conclusion Anopheles mosquitoes from Peninsular Malaysia show close evolutionary relationship with the Asian anophelines. Nevertheless, genetic differences due to geographical segregation can be seen. Meanwhile, some Anopheles mosquitoes in Peninsular Malaysia show vicariance, exemplified by the emergence of distinct cluster of An. sinensis population. PMID:24993022

  4. Nucleotide sequencing and analysis of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) of Taylorella equigenitalis, as an important pathogen for contagious equine metritis (CEM).

    PubMed

    Kagawa, S; Nagano, Y; Tazumi, A; Murayama, O; Millar, B C; Moore, J E; Matsuda, M

    2006-05-01

    The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184(T), Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184(T) and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184(T) and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.

  5. Improving Resident Knowledge of Spacers.

    PubMed

    Kilgore, Brian; Al Katranji, Khalid; Woodall, Meredith; Shepherd, Meagan; Flesher, Susan L

    2016-10-01

    Studies show the delivery of inhaled medications is maximized when a metered-dose inhaler (MDI) with a spacer is utilized. Our residents expressed concern with their knowledge of MDIs and spacers. This study was designed to address those concerns. Residents were given a 12-question pre-intervention, self-assessment questionnaire that explored their overall knowledge and comfort in utilizing MDI with spacers. Participants then received educational intervention via multimedia videos and a demonstration of proper use of MDI with spacer. Participants were given the same questionnaire immediately following the education and again 3 months later. Improvement was significant (P < .05) for each element studied as derived from the 12 questions. Improvement remained significant when these variables were assessed in the 3-month follow-up. In this study, we successfully improved the ability of our residents to deliver quality care by improving their knowledge and confidence in utilizing MDIs with spacers. © The Author(s) 2016.

  6. The calmodulin intergenic spacer as molecular target for characterization of Leishmania species

    PubMed Central

    2014-01-01

    Background Human leishmaniasis is a neglected disease caused by parasites of the genus Leishmania. Clinical aspects of this disease can vary significantly, reflecting the wide range of parasites in the genus Leishmania. Knowing accurately the Leishmania species infecting humans is important for clinical case management and evaluation of epidemiological risk. Calmodulin is an essential gene in trypanosomatids that modulates the calcium metabolism in various cellular activities. Despite its strong conservation in trypanosomatids, it has been recently observed that its untranslated regions (UTR) diverge among species. Methods In this study we analyzed the sequences and the absolute dinucleotide frequency of the intergenic spacer of the calmodulin gene (containing both, 3′ and 5′UTR) in nine reference Leishmania species and ten clinical isolates obtained from patients with cutaneous leishmaniasis. Results We show that the short calmodulin intergenic spacers exhibit features that make them interesting for applications in molecular characterization and phylogenetic studies of Leishmania. Dendrograms based on sequence alignments and on the dinucleotide frequency indicate that this particular region of calmodulin gene might be useful for species typing between the Leishmania and Viannia subgenera. Conclusions Mutations and composition of the calmodulin intergenic spacer from Leishmania species might have taxonomic value as parameters to define if an isolate is identical to a certain species or belongs to one of the two current subgenera. PMID:24438764

  7. Molecular phylogenetic studies on filarial parasites based on 5S ribosomal spacer sequences.

    PubMed

    Xie, H; Bain, O; Williams, S A

    1994-06-01

    This paper is the first large-scale molecular phylogenetic study on filarial parasites (family Onchocercidae) which includes 16 species of 6 genera: Brugia beaveri Ash et Little, 1962, B. buckleyi Dissanaike et Paramananthan, 1961; B. malayi (Brug, 1927) Buckley, 1960; B. pahangi (Buckley et Edeson, 1956) Buckley, 1960; B. patei (Buckley, Nelson et Heisch, 1958) Buckley, 1960; B. timori Partono et al, 1977; Wuchereria bancrofti (Cobbold, 1877) Seurat, 1921: W. kalimantani Palmieri. Purnomo, Dennis and Marwoto, 1980: Mansonella perstans (Manson, 1891) Eberhard et Orihel, 1984; loa loc, Stiles, 1905; Onchocerca volvulus (Leuckart, 1983) Railliet er Henry, 1910; O. ochengi Bwangamoi, 1969; O. gutturosa Neumann, 1910; Dirofilaria immitis (Leidy, 1856) Railliet e Henry, 1911; Acanthocheilonema viteae (Krepkogorskaya, 1933) Bain, Baker et Chabaud, 1982 and Litomosoides sigmodontis Chandler, 1931. 5S rRNA gene spacer region sequence data were collected by PCR, cloning and dideoxy sequencing. The 5S rRNA gene spacer region sequences were aligned and analyzed by maximum parsimony algorithms, distance methods and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the robustness of the different phylogenetic reconstructions. The data indicated that 5S spacer region sequences are highly conserved within species yet differ significantly between species. Spliced leader sequences were observed in all of the 5S rDNA spacers with no sequence variation, although flanking region sequence and length heterogeneity was observed even within species. All of the various tree-building methods gave very similar results. This study identified four clades which are strongly supported by bootstrap analysis the Brugia clade; the Wuchereria clade; the Brugia-Wuchereria clade and the Onchocerca clade. The analyses indicated that L. sigmodontis and A. viteae may be the most primitive among the 16 species studied. The data did not show any close

  8. The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

    SciTech Connect

    Luo, Michelle L.; Jackson, Ryan N.; Denny, Steven R.; Tokmina-Lukaszewska, Monika; Maksimchuk, Kenneth R.; Lin, Wayne; Bothner, Brian; Wiedenheft, Blake; Beisel, Chase L.

    2016-05-12

    Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Lastly, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.

  9. Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli.

    PubMed

    Johnson, Y A; Nagpal, M; Krahmer, M T; Fox, K F; Fox, A

    2000-05-01

    Assessment of 16S-23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5' terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S-23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.

  10. Trypanosoma cruzi I genotypes in different geographic regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced leader genes✯

    PubMed Central

    Cura, Carolina I.; Mejía-Jaramillo, Ana M.; Duffy, Tomás; Burgos, Juan M.; Rodriguero, Marcela; Cardinal, Marta V.; Kjos, Sonia; Gurgel-Gonçalves, Rodrigo; Blanchet, Denis; De Pablos, Luis M.; Tomasini, Nicolás; Silva, Alex Da; Russomando, Graciela; Cuba Cuba, Cesar A.; Aznar, Christine; Abate, Teresa; Levin, Mariano J.; Osuna, Antonio; Gürtler, Ricardo E.; Diosque, Patricio; Solari, Aldo; Triana-Chávez, Omar; Schijman, Alejandro G.

    2011-01-01

    The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harboring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia + Tc Id, Tc Ia + Tc Ie and Tc Id + Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time. PMID:20670628

  11. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food.

    PubMed

    Chen, Peng; Zhao, Yang; Wu, Zhengrong; Liu, Ronghui; Xu, Ruixiang; Yan, Lei; Li, Hongyu

    2016-03-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411.

  12. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food

    PubMed Central

    Chen, Peng; Zhao, Yang; Wu, Zhengrong; Liu, Ronghui; Xu, Ruixiang; Yan, Lei; Li, Hongyu

    2015-01-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411. PMID:26981389

  13. Differential utilization of T cell receptor TCR alpha/TCR delta locus variable region gene segments is mediated by accessibility.

    PubMed

    Lee, Yu Nee; Alt, Frederick W; Reyes, Julia; Gleason, Megan; Zarrin, Ali A; Jung, David

    2009-10-13

    T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRdelta locus is contained within the TCRalpha locus; TCRalpha variable region exons are encoded by TRAV and TRAJ segments and those of TCRdelta by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5'TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development.

  14. Differential utilization of T cell receptor TCRα/TCRδ locus variable region gene segments is mediated by accessibility

    PubMed Central

    Lee, Yu Nee; Alt, Frederick W.; Reyes, Julia; Gleason, Megan; Zarrin, Ali A.; Jung, David

    2009-01-01

    T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRδ locus is contained within the TCRα locus; TCRα variable region exons are encoded by TRAV and TRAJ segments and those of TCRδ by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5′TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development. PMID:19805067

  15. Regional Variation in the Density of Essential Genes in Mice

    PubMed Central

    Hentges, Kathryn E; Pollock, David D; Liu, Bin; Justice, Monica J

    2007-01-01

    In most species, and particularly in vertebrates, the percentage of genes absolutely required for survival, the essential genes, has not been estimated. To obtain this estimation, we used the mouse as an experimental model to carry out high-efficiency N-ethyl-N-nitrosourea (ENU) mutagenesis screens in two balancer chromosome regions, and compared our results to a third previously published screen. The number of essential genes in each region was predicted based on allele frequencies. We determined that the density of essential genes differs by up to an order of magnitude among genomic regions. This indicates that extrapolating from regional estimates to genome-wide estimates of essential genes has a huge variance. A particularly high density of essential genes on mouse Chromosome 11 coincides with a high degree of regional linkage conservation, providing a possible causal explanation for the density variation. This is the first demonstration of regional variation in essential gene density in the mouse genome. PMID:17480122

  16. Chasing the hare - evaluating the phylogenetic utility of a nuclear single copy gene region at and below species level within the species rich group Peperomia (Piperaceae).

    PubMed

    Naumann, Julia; Symmank, Lars; Samain, Marie-Stéphanie; Müller, Kai F; Neinhuis, Christoph; dePamphilis, Claude W; Wanke, Stefan

    2011-12-12

    The rapidly increasing number of available plant genomes opens up almost unlimited prospects for biology in general and molecular phylogenetics in particular. A recent study took advantage of this data and identified a set of nuclear genes that occur in single copy in multiple sequenced angiosperms. The present study is the first to apply genomic sequence of one of these low copy genes, agt1, as a phylogenetic marker for species-level phylogenetics. Its utility is compared to the performance of several coding and non-coding chloroplast loci that have been suggested as most applicable for this taxonomic level. As a model group, we chose Tildenia, a subgenus of Peperomia (Piperaceae), one of the largest plant genera. Relationships are particularly difficult to resolve within these species rich groups due to low levels of polymorphisms and fast or recent radiation. Therefore, Tildenia is a perfect test case for applying new phylogenetic tools. We show that the nuclear marker agt1, and in particular the agt1 introns, provide a significantly increased phylogenetic signal compared to chloroplast markers commonly used for low level phylogenetics. 25% of aligned characters from agt1 intron sequence are parsimony informative. In comparison, the introns and spacer of several common chloroplast markers (trnK intron, trnK-psbA spacer, ndhF-rpl32 spacer, rpl32-trnL spacer, psbA-trnH spacer) provide less than 10% parsimony informative characters. The agt1 dataset provides a deeper resolution than the chloroplast markers in Tildenia. Single (or very low) copy nuclear genes are of immense value in plant phylogenetics. Compared to other nuclear genes that are members of gene families of all sizes, lab effort, such as cloning, can be kept to a minimum. They also provide regions with different phylogenetic content deriving from coding and non-coding parts of different length. Thus, they can be applied to a wide range of taxonomic levels from family down to population level. As more

  17. Widespread transcription of a Qa region gene in adult mice

    PubMed Central

    1987-01-01

    The mouse MHC class I family includes genes encoded in four regions: H- 2K, H-2D, Qa and Tla. While K/D genes are well characterized, relatively little is known about Qa or Tla genes. We have studied the transcription of a B10.P Qa region gene. DNA sequence comparisons of the transmembrane region, supported by Southern blot analysis of cosmid and genomic DNAs from BALB/c and C57BL/10, demonstrate the lambda 3a gene corresponds to Q4p. In both Northern blots and RNA protection experiments using probes derived from the 3' noncoding region, we found that Q4, like the H-2K and H-2D genes, is widely transcribed in B10.P tissues. These data demonstrate for the first time widespread transcription of a Qa gene. PMID:2439640

  18. Identification of Brucella by Ribosomal-Spacer-Region PCR and Differentiation of Brucella canis from Other Brucella spp. Pathogenic for Humans by Carbohydrate Profiles

    PubMed Central

    Fox, Karen F.; Fox, Alvin; Nagpal, Madan; Steinberg, Paul; Heroux, Karen

    1998-01-01

    Molecular and chemical characteristics often provide complementary information in the differentiation of closely related organisms. The genus Brucella consists of a highly conserved group of organisms. Identification of the four species pathogenic in humans (Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis) is problematic for many clinical laboratories that depend primarily on serology and phenotypic characteristics to differentiate species. PCR amplification of the 16S-23S ribosomal DNA interspace region was evaluated for species-specific polymorphism. B. abortus, B. melitensis, B. suis, and B. canis produced identical PCR interspace profiles. However, these PCR products were unique to brucellae, allowing them to be readily distinguished from other gram-negative bacteria (including Bartonella spp. and Agrobacterium spp.). Carbohydrate profiles differentiated B. canis from the other three Brucella species due to the absence of the rare amino sugar quinovosamine in the three other species. PCR of the rRNA interspace region is useful in identification of the genus Brucella, while carbohydrate profiling is capable of differentiating B. canis from the other Brucella species. PMID:9774568

  19. Demarcating the gene-rich regions of the wheat genome

    PubMed Central

    Erayman, Mustafa; Sandhu, Devinder; Sidhu, Deepak; Dilbirligi, Muharrem; Baenziger, P. S.; Gill, Kulvinder S.

    2004-01-01

    By physically mapping 3025 loci including 252 phenotypically characterized genes and 17 quantitative trait loci (QTLs) relative to 334 deletion breakpoints, we localized the gene-containing fraction to 29% of the wheat genome present as 18 major and 30 minor gene-rich regions (GRRs). The GRRs varied both in gene number and density. The five largest GRRs physically spanning <3% of the genome contained 26% of the wheat genes. Approximate size of the GRRs ranged from 3 to 71 Mb. Recombination mainly occurred in the GRRs. Various GRRs varied as much as 128-fold for gene density and 140-fold for recombination rates. Except for a general suppression in 25–40% of the chromosomal region around centromeres, no correlation of recombination was observed with the gene density, the size, or chromosomal location of GRRs. More than 30% of the wheat genes are in recombination-poor regions thus are inaccessible to map-based cloning. PMID:15240829

  20. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

    PubMed Central

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-01-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories. PMID:27074256

  1. Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin.

    PubMed

    Gupta, K N; Baranwal, V K; Haq, Q M R

    2012-03-01

    High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5-8 years old orchards of kinnow mandarin {Citrus reticulate Balanco ('King' × 'Willow mandarin')} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus ('Ca L. asiaticus') showing maximum identity of 95.9% with 'Ca L. asiaticus' from USA and Brazil in 16S rRNA. The study indicates definite association of 'Ca L. asiaticus' with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.

  2. Differentiation of Acinetobacter baumannii biotypes by amplification of 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Garcia, A; Montoya, R; Bello, H; Gonzalez, G; Dominguez, M; Zemelman, R

    1996-01-01

    Isolates of Acinetobacter baumannii (32 strains) from blood samples obtained from patients in five Chilean hospitals were identified and biotyped according to their phenotypic properties. They were also submitted to random amplified polymorphic DNA (RAPD) using eight randomly designed 10-mers and the core sequence of M13 phage (15-mers) as well as amplification of the spacer regions between 16S and 23S genes in the prokaryotic rRNA genetic loci. With some primers, RAPD discriminated between biotypes, whereas with others each isolate showed a particular profile. When amplification of spacer regions was performed, a clear correlation between patterns and biotypes was found. This last technique allowed correct biotyping of clinical isolates. Both genetic methods might be used for the identification of A. baumannii biotypes.

  3. Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability

    PubMed Central

    Ranjard, L.; Poly, F.; Lata, J.-C.; Mougel, C.; Thioulouse, J.; Nazaret, S.

    2001-01-01

    Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi. PMID:11571146

  4. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

    PubMed Central

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-01-01

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

  5. Analysis of the genetic diversity of Legionella by sequencing the 23S-5S ribosomal intergenic spacer region: from phylogeny to direct identification of isolates at the species level from clinical specimens.

    PubMed

    Grattard, Florence; Ginevra, Christophe; Riffard, Serge; Ros, Alain; Jarraud, Sophie; Etienne, Jerome; Pozzetto, Bruno

    2006-01-01

    This study focuses on the interest of the hypervariable 23S-5S ribosomal intergenic spacer region (ISR) of the genus Legionella to analyze the phylogenic diversity of Legionella at the species and subspecies levels and to identify isolates directly from clinical specimens. The method, using a real-time PCR assay with a single primer pair followed by sequencing, was able to identify correctly 49 reference strains of Legionella belonging to 37 different species, including those implicated in human infections, and to clearly differentiate the three subspecies of L. pneumophila. Based on sequence similarities, the 23S-5S ISR sequences were much more variable than the rpoB and mip sequences (P<0.0001 by the Wilcoxon signed rank test). The 23S-5S ISR method was able to cluster Legionella species in accordance with phenotypic traits, such as autofluorescence or fatty acid membrane composition. Using maximum parsimony methods, the rpoB and 23S-5S ISR data sets were shown to be incongruent (P<0.001). In contrast, the 23S-5S ISR and the mip data sets were found to be congruent (P=0.313), suggesting the interest of combining these two regions to demonstrate phylogenetic links between Legionella species. This molecular assay was shown able to both detect Legionella DNA directly in respiratory specimens from patients exhibiting a Legionella infection and provide accurate identification of the bacterium at the species level in the tested specimens. These properties open a wide range of applications to the 23S-5S ISR sequencing method, from taxonomic analyses to clinical and epidemiological investigations.

  6. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

    PubMed

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-09-29

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

  7. Comparison of Virulence Gene Identification, Ribosomal Spacer PCR, and Pulsed-Field Gel Electrophoresis for Typing of Staphylococcus aureus Strains Isolated from Cases of Subclinical Bovine Mastitis in the United States.

    PubMed

    Adkins, Pamela R F; Middleton, John R; Fox, Lawrence K

    2016-07-01

    Staphylococcus aureus is one of the most important pathogens causing contagious mastitis in dairy cattle worldwide. The objectives of this study were to determine if recently described S. aureus genotype B was present among previously characterized isolates from cases of bovine intramammary infection in the United States and to compare pulsed-field gel electrophoresis (PFGE) to the combination of ribosomal spacer PCR (RS-PCR) and virulence gene identification for typing of S. aureus strains. The hypothesis was that isolates that were previously characterized as contagious would be identified as genotype B and that the results of the two strain-typing methods would be comparable. Isolates were selected from a collection of S. aureus isolates from eight dairy farms. Mammary quarter milk somatic cell count (SCC) and N-acetyl-β-d-gluconaminidase (NAGase) activity data were known and used to evaluate strain pathogenicity. RS-PCR was performed with conventional gel electrophoresis, and PCR was used for toxin gene identification. RS-PCR patterns were associated with a specific virulence gene pattern, as previously reported. Five RS-PCR banding patterns were identified. None of the isolates were characterized as genotype B. No association between RS-PCR types and milk SCC was found; however, NAGase activity was significantly higher in milk from mammary glands infected with RS-PCR banding type 1 (RSP type 1) than in milk from those infected with RSP type 2. The discriminatory power values were 1.0 and 0.46 for PFGE and RS-PCR, respectively. These data suggest that genotype B may have a limited geographic distribution and that PFGE is more discriminatory than RS-PCR performed with conventional gel electrophoresis for typing of S. aureus isolates of bovine origin. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, β-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Tam, Emily W T; Chen, Jonathan H K; Lau, Eunice C L; Ngan, Antonio H Y; Fung, Kitty S C; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2014-04-01

    Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.

  9. The CRISPR Spacer Space Is Dominated by Sequences from Species-Specific Mobilomes

    PubMed Central

    Shmakov, Sergey A.; Sitnik, Vassilii; Makarova, Kira S.; Wolf, Yuri I.; Severinov, Konstantin V.

    2017-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems store the memory of past encounters with foreign DNA in unique spacers that are inserted between direct repeats in CRISPR arrays. For only a small fraction of the spacers, homologous sequences, called protospacers, are detectable in viral, plasmid, and microbial genomes. The rest of the spacers remain the CRISPR “dark matter.” We performed a comprehensive analysis of the spacers from all CRISPR-cas loci identified in bacterial and archaeal genomes, and we found that, depending on the CRISPR-Cas subtype and the prokaryotic phylum, protospacers were detectable for 1% to about 19% of the spacers (~7% global average). Among the detected protospacers, the majority, typically 80 to 90%, originated from viral genomes, including proviruses, and among the rest, the most common source was genes that are integrated into microbial chromosomes but are involved in plasmid conjugation or replication. Thus, almost all spacers with identifiable protospacers target mobile genetic elements (MGE). The GC content, as well as dinucleotide and tetranucleotide compositions, of microbial genomes, their spacer complements, and the cognate viral genomes showed a nearly perfect correlation and were almost identical. Given the near absence of self-targeting spacers, these findings are most compatible with the possibility that the spacers, including the dark matter, are derived almost completely from the species-specific microbial mobilomes. PMID:28928211

  10. Generator stator core vent duct spacer posts

    DOEpatents

    Griffith, John Wesley; Tong, Wei

    2003-06-24

    Generator stator cores are constructed by stacking many layers of magnetic laminations. Ventilation ducts may be inserted between these layers by inserting spacers into the core stack. The ventilation ducts allow for the passage of cooling gas through the core during operation. The spacers or spacer posts are positioned between groups of the magnetic laminations to define the ventilation ducts. The spacer posts are secured with longitudinal axes thereof substantially parallel to the core axis. With this structure, core tightness can be assured while maximizing ventilation duct cross section for gas flow and minimizing magnetic loss in the spacers.

  11. Spacer grid assembly and locking mechanism

    DOEpatents

    Snyder, Jr., Harold J.; Veca, Anthony R.; Donck, Harry A.

    1982-01-01

    A spacer grid assembly is disclosed for retaining a plurality of fuel rods in substantially parallel spaced relation, the spacer grids being formed with rhombic openings defining contact means for engaging from one to four fuel rods arranged in each opening, the spacer grids being of symmetric configuration with their rhombic openings being asymmetrically offset to permit inversion and relative rotation of the similar spacer grids for improved support of the fuel rods. An improved locking mechanism includes tie bars having chordal surfaces to facilitate their installation in slotted circular openings of the spacer grids, the tie rods being rotatable into locking engagement with the slotted openings.

  12. Use of PCR primers and probes based on the 23S rRNA and internal transcription spacer (ITS) gene sequence for the detection and enumerization of Lactobacillus acidophilus and Lactobacillus plantarum in feed supplements.

    PubMed

    Tsai, Cheng-Chih; Lai, Chieh-Hsien; Yu, Bi; Tsen, Hau-Yang

    2010-06-01

    Novel polymerase chain reaction (PCR) primers designed from the 16S-23S internal transcription spacer (ITS) rRNA and 23S rRNA genes, respectively, were used for the specific detection of Lactobacillus acidophilus and Lactobacillus plantarum. Molecular weights of the PCR products were 221 and 599 bp, respectively. Strains of L. acidophilus and L. plantarum obtained from the culture center, dairy products, infant stool and other samples, could be identified with these PCR primers. DNAs from other lactic acid bacteria (LAB) species including strains of Lactobacillus pentosus which was closely related to L. plantarum, and bacteria species other than LAB, would not generate the false positive results. When this PCR primer set was used for the detection of L. acidophilus and L. plantarum in feed supplement or feed starter samples, reliable results were obtained. Furthermore, when these L. acidophilus or L. plantarum specific primers were used as DNA probes for the colony hybridization of L. acidophilus and L. plantarum, the viable cells of these LAB species in culture and feed supplements or starter products could be identified and enumerized. The method described here thus offers a rapid and economic way to inspect and assure the quality of the feed supplements or fermentation starters.

  13. Molecular characterization of ribosomal intergenic spacer in the tadpole shrimp Triops cancriformis (Crustacea, Branchiopoda, Notostraca).

    PubMed

    Luchetti, Andrea; Scanabissi, Franca; Mantovani, Barbara

    2006-08-01

    Nuclear ribosomal DNA constitutes a multigene family, with tandemly arranged units linked by an intergenic spacer (IGS), which contains initiation/termination transcription signals and usually tandemly arranged subrepeats. The structure and variability of the IGS region are analyzed here in hermaphroditic and parthenogenetic populations of the "living fossil" Triops cancriformis (Branchiopoda, Notostraca). The results indicate the presence of concerted evolution at the population level for this G+C-rich IGS region as a whole, with the major amount of genetic variability found outside the subrepeat region. The subrepeats region is composed of 3 complete repeats (a, c, d) intermingled with 3 repeat fragments (b, e, f) and unrelated sequences. The most striking datum is the absolute identity of subrepeats (except type d) occupying the same position in different individuals/populations. A putative promoter sequence is present upstream of the 18S rRNA gene, but not in subrepeats, which is at variance with other arthropod IGSs. The absence of a promoter sequence in the subrepeats and subrepeat sequence conservation suggests that this region acts as an enhancer simply by its repetitive nature, as observed in some vertebrates. The putative external transcribed spacer (840 bp) shows hairpin structures, as in yeasts, protozoans, Drosophila, and vertebrates.

  14. Development of high performance BWR spacer

    SciTech Connect

    Morooka, Shinichi; Shirakawa, Kenetu; Mitutake, Tohru; Yamamoto, Yasushi; Yano, Takashi; Kimura, Jiro

    1996-07-01

    The spacer has a significant effect on thermal hydraulic performance of BWR fuel assembly. The purpose of this study is to develop a new BWR spacer with high critical power and low pressure drop performance. The developed high performance spacer is a ferrule type spacer with twisted tape and improved flow tab. This spacer is called CYCLONE spacer. Critical power and pressure drop have been measured at BEST (BWR Experimental Loop for Stability and Transient test) of Toshiba Corporation. The test bundle consists of electrically heated rods in a 4x4 array configuration. These heater rods are indirectly heated. The heated length and outer diameter of the heater rod, as well as the number and the axial locations of the spacers, are the same as for those for a BWR fuel assembly. The axial power shape is stepped cosine (1.4 of the maximum peaking factor). Two test assemblies with different radial power distribution have been used. One test assembly has the maximum power rods at the center of the test assembly and the other has the maximum power rods near the channel wall. The results show that the critical power performance of CYCLONE spacer is 10 to 25 % higher than that of the ferrule spacers, while the pressure drop for CYCLONE spacer is nearly equal to that of the ferrule spacer.

  15. CRISPR interference and priming varies with individual spacer sequences.

    PubMed

    Xue, Chaoyou; Seetharam, Arun S; Musharova, Olga; Severinov, Konstantin; Brouns, Stan J J; Severin, Andrew J; Sashital, Dipali G

    2015-12-15

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naïve' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.

  16. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria

    PubMed Central

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J.; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I–V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC. PMID:27749897

  17. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

    PubMed

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

  18. [Interspecific hybridization in the genus Paeonia (Paeoniaceae): polymorphic sites in transcribed spacers of the 45S rRNA genes as indicators of natural and artificial peony hybrids].

    PubMed

    Punina, E O; Machs, E M; Krapivskaia, E E; Kim, E S; Mordak, E V; Miakoshina, Iu A; Rodionov, A V

    2012-07-01

    The ITS1-5.8S rDNA-ITS2 regions of 33 accessions belonging to 16 species and five natural and garden interspecific hybrids of the genus Paeonia L. were sequenced. Chromatograms of the peony hybrids demonstrated the presence of the signals, corresponding to two different nucleotides at the positions differing in the parents, indicating that in the hybrids, no rDNA isogenization usually occurred, and they preserved rDNA of both parents. Analysis of these polymorphic sites (PS) showed that P. x majkoae was interspecific hybrid between P. tenuifolia and P. caucasica. The ITS of P. hybrida differs from ITS of P. x majkoae in 19 mutations. Because of this, P. x majkoae is definitely not synonymous to P. hybrida. Comparative analysis of ITS 1-5.8S rDNA-ITS2 showed that species diversity in section Paeonia was based on recombination as a result of intraspecific hybridization of three haplotype families. Specifically, haplotypes A, typical of the P. tenuifolia and P. anomala genomes, haplotypes B, typical of P. mlokosewitschii and P. obovata, and haplotypes of family C, currently represented in rDNA of diploid and tetraploid forms of some Caucasian and Mediterranean species. The ITS regions many diploid peonies contain no dimorphic sites, while P. oreogeton, P. cambessedesii, P. rhodia, and P. daurica carry from ten to 17 PS, and supposed to be the interspecific hybrids. Most of the tetraploid peonies contain from six to 18 PS in the ITS regions. These are alloploids with one of the parental genomes similar to that of P. mlokosewitschii (B1), or P. obovata (B3). The second parental genome in P. banatica, P. peregrina, and P. russii is represented by the genome, close to that of P. tenuifolia (A). P. macrophylla, P. mascula, P. coriacea, P. wittmanniana, and P. tomentosa carry genome of series B and genome of series C, which slightly resembles genome A.

  19. Genes in one megabase of the HLA class I region

    SciTech Connect

    Wei, H.; Fan, Wu-Fang; Xu, Hongxia; Shukla, H.; Weissman, S.M. ); Parimoo, S. R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ ); Chaplin, D.D. )

    1993-11-15

    To define the gene content of the HLA class I region, cDNA selection was applied to three overlapping yeast artificial chromosomes (YACs) that spanned 1 megabase (Mb) of this region of the human major histocompatibility complex. These YACs extended from the region centromeric to HLA-E to the region telomeric to HLA-F. In additions to the recognized class I genes and pseudogenes and the anonymous non-class-I genes described recently by the authors and others, 20 additional anonymous cDNA clones were identified from this 1-Mb region. They also identified a long repetitive DNA element in the region between HLA-B and HLA-E. Homologues of this outside of the HLA complex. The portion of the HLA class I region represented by these YACs shows an average gene density as high as the class II and class III regions. Thus, the high gene density portion of the HLA complex is extended to more than 3 Mb.

  20. Sequence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species.

    PubMed

    Aktas, Münir; Bendele, Kylie G; Altay, Kürsat; Dumanli, Nazir; Tsuji, Masayoshi; Holman, Patricia J

    2007-07-20

    The genomic region spanning the two ribosomal RNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was cloned and sequenced from sixteen Theileria isolates. Each Theileria species possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among ruminant Theileria species. The spacers were most polymorphic in the agent of tropical theileriosis, Theileria annulata, and were more conserved in two benign species, Theileria buffeli and Theileria sergenti Chitose. Phylogenetic analysis of the rDNA ITS1-5.8S rRNA gene-ITS2 region clearly separated each taxon, placing them in three clusters. One held T. annulata, Theileria parva, and Theileria mutans, with the latter two most closely related. The second held T. sergenti Ikeda, T. sergenti Chitose, and T. buffeli, with the latter two most closely related. The third cluster held the Theileria ovis isolates.

  1. Identification of wheat chromosomal regions containing expressed resistance genes.

    PubMed Central

    Dilbirligi, Muharrem; Erayman, Mustafa; Sandhu, Devinder; Sidhu, Deepak; Gill, Kulvinder S

    2004-01-01

    The objectives of this study were to isolate and physically localize expressed resistance (R) genes on wheat chromosomes. Irrespective of the host or pest type, most of the 46 cloned R genes from 12 plant species share a strong sequence similarity, especially for protein domains and motifs. By utilizing this structural similarity to perform modified RNA fingerprinting and data mining, we identified 184 putative expressed R genes of wheat. These include 87 NB/LRR types, 16 receptor-like kinases, and 13 Pto-like kinases. The remaining were seven Hm1 and two Hs1(pro-1) homologs, 17 pathogenicity related, and 42 unique NB/kinases. About 76% of the expressed R-gene candidates were rare transcripts, including 42 novel sequences. Physical mapping of 121 candidate R-gene sequences using 339 deletion lines localized 310 loci to 26 chromosomal regions encompassing approximately 16% of the wheat genome. Five major R-gene clusters that spanned only approximately 3% of the wheat genome but contained approximately 47% of the candidate R genes were observed. Comparative mapping localized 91% (82 of 90) of the phenotypically characterized R genes to 18 regions where 118 of the R-gene sequences mapped. PMID:15020436

  2. Non-HLA region genes in insulin dependent diabetes mellitus.

    PubMed

    Field, L L

    1991-09-01

    The focus of this chapter is on the contribution of genes outside the HLA region to insulin dependent diabetes mellitus (IDDM) susceptibility. We review laboratory evidence for such genes from published studies and also present unpublished data from our recent research. The existence of genes predisposing to IDDM in the region of the insulin (INS) gene now appears established. Association analysis has demonstrated an increased frequency of class 1 alleles of the 5' INS polymorphism in diabetics compared with controls, and a new method of analysis (AFBAC) has shown that this association is not an artefact of population stratification. Interestingly, the effect of INS region susceptibility on IDDM cannot be detected by linkage analysis, suggesting that if a genetic marker locus is close to a disease susceptibility locus, association analysis may be a more sensitive method than linkage analysis for detecting the susceptibility locus. There is no convincing evidence that genes in the T cell receptor beta chain (TCRB) or alpha chain (TCRA) regions influence predisposition to IDDM, either directly, or indirectly through interaction with HLA region genes. However, we present new evidence for interaction between TCRB and immunoglobulin heavy chain (Gm) region genes in IDDM: diabetics who are positive for the IgG2 allotype G2m(23) have significantly different frequencies of a TCRB restriction fragment length polymorphism (RFLP) than those who are negative for the allotype. Gm region genes also appear to have indirect effects on IDDM susceptibility through interaction with HLA and INS region genes: DR3/4 and non-DR3/4 diabetics have significantly different frequencies of G2m(23), and INS1/1 and non-INS1/1 diabetics also have significantly different frequencies of this allotype. To our knowledge, there are no other studies of Gm-TCRB or Gm-INS interaction in IDDM susceptibility. Evidence for Gm-HLA interaction in IDDM has been published by several other groups of

  3. Evidence for gene conversion among immunoglobulin heavy chain variable region genes.

    PubMed

    Clarke, S H; Rudikoff, S

    1984-03-01

    We have previously reported that the VH region amino acid sequence of a phosphocholine (PC)-binding hybridoma antibody of CBA/J origin, HP101 6G6 (6G6), differs extensively from the VH regions of other PC-binding antibodies. The sequence of 6G6 VH appears to be derived from a gene homologous to the BALB/c V11 gene, a member of the PC VH (T15 VH) gene family not normally used to encode PC-binding antibodies. The 6G6 VH sequence differs from the translated sequence of V11 by six amino acids, four of which occur at the same position in other members of this gene family. This coincidence led to the proposal that the 6G6 VH gene was derived by gene conversion involving three genes of the PC VH gene family. We report here the nucleic acid sequence of the rearranged VH gene of hybridoma 6G6. This sequence supports our previous suggestion of gene conversion by confirming those differences, relative to the BALB/c V11 gene sequence, that are encoded by other members of this gene family, and extends this correlation to include three silent base pair substitutions as well. In addition, 5' noncoding region sequence and Southern blot analysis using probes derived from the coding and 5' noncoding regions confirm that the 6G6 VH gene is likely to be derived from the V11 homologue in CBA/J mice, and suggest that all three genes believed to be involved in the generation of the 6G6 VH gene are present in the CBA/J genome, a prerequisite for their involvement in gene conversion.

  4. The regions of sequence variation in caulimovirus gene VI.

    PubMed

    Sanger, M; Daubert, S; Goodman, R M

    1991-06-01

    The sequence of gene VI from figwort mosaic virus (FMV) clone x4 was determined and compared with that previously published for FMV clone DxS. Both clones originated from the same virus isolation, but the virus used to clone DxS was propagated extensively in a host of a different family prior to cloning whereas that used to clone x4 was not. Differences in the amino acid sequence inferred from the DNA sequences occurred in two clusters. An N-terminal conserved region preceded two regions of variation separated by a central conserved region. Variation in cauliflower mosaic virus (CaMV) gene VI sequences, all of which were derived from virus isolates from hosts from one host family, was similar to that seen in the FMV comparison, though the extent of variation was less. Alignment of gene VI domains from FMV and CaMV revealed regions of amino acid sequence identical in both viruses within the conserved regions. The similarity in the pattern of conserved and variable domains of these two viruses suggests common host-interactive functions in caulimovirus gene VI homologues, and possibly an analogy between caulimoviruses and certain animal viruses in the influence of the host on sequence variability of viral genes.

  5. Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E. coli K-12.

    PubMed

    García-Martínez, J; Martínez-Murcia, A; Antón, A I; Rodríguez-Valera, F

    1996-11-01

    Several 16S to 23S spacers of 354 bp have been sequenced from six Escherichia coli strains belonging to the ECOR collection. Four phylogenetically informative variable sites were identified. The results of their comparison confirm the existence of two major phylogenetic branches in this species, as previously reported. Remarkable intercistronic heterogeneity was found in strain ECOR35 and its closest relatives, in which at least one of the operons has suffered a major mutagenic event or has an independent phylogenetic origin.

  6. The chloroplast trnT-trnF region in the seed plant lineage Gnetales.

    PubMed

    Won, Hyosig; Renner, Susanne S

    2005-10-01

    The trnT-trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL intron, a group I intron, and the trnT-trnL and trnL-trnF intergenic spacers. We analyzed the evolution of the region in the three genera of the gymnosperm lineage Gnetales (Gnetum, Welwitschia, and Ephedra), with especially dense sampling in Gnetum for which we sequenced 41 accessions, representing most of the 25-35 species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, while the spacers are so variable in length and composition that homology cannot be found even among the three genera. Palindromic sequences that form hairpin structures were detected in the trnL-trnF spacer, but neither spacer contained promoter elements for the tRNA genes. The absence of promoters, presence of hairpin structures in the trnL-trnF spacer, and high sequence variation in both spacers together suggest that trnT and trnF are independently transcribed. Our model for the expression and processing of the genes tRNA(Thr)(UGU), tRNA(Leu)(UAA), and tRNA(Phe) (GAA) therefore attributes the seemingly neutral evolution of the two spacers to their escape from functional constraints.

  7. CRISPR interference and priming varies with individual spacer sequences

    PubMed Central

    Xue, Chaoyou; Seetharam, Arun S.; Musharova, Olga; Severinov, Konstantin; J. Brouns, Stan J.; Severin, Andrew J.; Sashital, Dipali G.

    2015-01-01

    CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring ‘spacer’ sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid ‘primed’ adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR–Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed ‘naïve’ adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection. PMID:26586800

  8. Identification of genes from the Treacher Collins candidate region

    SciTech Connect

    Dixon, M.; Dixon, J.; Edwards, S. |

    1994-09-01

    Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development. The TCOF1 locus has previously been mapped to chromosome 5q32-33. The candidate gene region has been defined as being between two flanking markers, ribosomal protein S14 (RPS14) and Annexin 6 (ANX6), by analyzing recombination events in affected individuals. It is estimated that the distance between these flanking markers is 500 kb by three separate analysis methods: (1) radiation hybrid mapping; (2) genetic linkage; and (3) YAC contig analysis. A cosmid contig which spans the candidate gene region for TCOF1 has been constructed by screening the Los Alamos National Laboratory flow-sorted chromosome 5 cosmid library. Cosmids were obtained by using a combination of probes generated from YAC end clones, Alu-PCR fragments from YACs, and asymmetric PCR fragments from both T7 and T3 cosmid ends. Exon amplifications, the selection of genomic coding sequences based upon the presence of functional splice acceptor and donor sites, was used to identify potential exon sequences. Sequences found to be conserved between species were then used to screen cDNA libraries in order to identify candidate genes. To date, four different cDNAs have been isolated from this region and are being analyzed as potential candidate genes for TCOF1. These include the genes encoding plasma glutathione peroxidase (GPX3), heparin sulfate sulfotransferase (HSST), a gene with homology to the ETS family of proteins and one which shows no homology to any known genes. Work is also in progress to identify and characterize additional cDNAs from the candidate gene region.

  9. The CRISPR Spacer Space Is Dominated by Sequences from Species-Specific Mobilomes.

    PubMed

    Shmakov, Sergey A; Sitnik, Vassilii; Makarova, Kira S; Wolf, Yuri I; Severinov, Konstantin V; Koonin, Eugene V

    2017-09-19

    Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems store the memory of past encounters with foreign DNA in unique spacers that are inserted between direct repeats in CRISPR arrays. For only a small fraction of the spacers, homologous sequences, called protospacers, are detectable in viral, plasmid, and microbial genomes. The rest of the spacers remain the CRISPR "dark matter." We performed a comprehensive analysis of the spacers from all CRISPR-cas loci identified in bacterial and archaeal genomes, and we found that, depending on the CRISPR-Cas subtype and the prokaryotic phylum, protospacers were detectable for 1% to about 19% of the spacers (~7% global average). Among the detected protospacers, the majority, typically 80 to 90%, originated from viral genomes, including proviruses, and among the rest, the most common source was genes that are integrated into microbial chromosomes but are involved in plasmid conjugation or replication. Thus, almost all spacers with identifiable protospacers target mobile genetic elements (MGE). The GC content, as well as dinucleotide and tetranucleotide compositions, of microbial genomes, their spacer complements, and the cognate viral genomes showed a nearly perfect correlation and were almost identical. Given the near absence of self-targeting spacers, these findings are most compatible with the possibility that the spacers, including the dark matter, are derived almost completely from the species-specific microbial mobilomes.IMPORTANCE The principal function of CRISPR-Cas systems is thought to be protection of bacteria and archaea against viruses and other parasitic genetic elements. The CRISPR defense function is mediated by sequences from parasitic elements, known as spacers, that are inserted into CRISPR arrays and then transcribed and employed as guides to identify and inactivate the cognate parasitic genomes. However, only a small fraction of the CRISPR spacers match

  10. Randomized trial of spacers in asthma.

    PubMed

    Dahiya, Baljit; Mathew, Joseph L; Singh, Meenu

    2007-07-01

    To compare the efficacy of all types of spacers commonly available to children in India. 150 children 5-14 yr of age with persistent asthma presenting with peak expiratory flow (PEF) < 70% of personal best were randomized to receive 200 mg salbutamol through one of five spacers: A) 750 ml spacer with valve, B) 165 ml spacer with valve, C) 250 ml spacer without valve, D) 1000 ml indigenously made spacer without valve and E) 500 ml indigenously made spacer without valve. PEF measurement was repeated 15 minutes later. Children> 8 yr old performed spirometry in addition to PEF. Absolute change and percentage improvement of PEF and FEV1 were compared among the groups. Subjects in all groups had comparable baseline demographic characteristics and PEF. All showed significant improvement in PEF and FEV1 over baseline values. The change in PEF and percentage improvement were comparable among all five groups (p=0.780 and p=0.955 respectively). Likewise change in FEV1 and percentage improvement were also comparable. The five groups showed no difference in efficacy, irrespective of severity of baseline airway obstruction. The five spacers were equally efficacious for the delivery of bronchodilator in children with moderate persistent asthma presenting with airway obstruction.

  11. Identification of candidate genes in the spinal muscular atrophy gene region

    SciTech Connect

    Carter, T.A.; Wang, C.H.; Vitale, E.

    1994-09-01

    The SMA disease gene region on chromosome 5q13 has been characterized by the presence of low copy repeat element DNA and concomitant genomic instability. The identification of candidate genes has been complicated by the low-copy repeat sequences which include coding and non-coding DNA. To circumvent this problem, we have focused on the identification and characterization of exonic DNA sequence isolated from YAC-derived bacteriophage subclones. Contrary to hybridization-based protocols which identify all closely homologous genes, exons isolated in this manner are known to map the disease gene region. Exon trapping was limited to an approximately 400 kb {open_quotes}minimum genetic region{close_quotes} defined by recombination mapping. Approximately six new genes have been identified by this method. One of the candidate genes is expressed predominantly in muscle and demonstrates sequence homology with known translational regulatory proteins. Physical mapping places the gene within the minimal genetic region and adjacent to a region characterized with genomic instability. We will report our progress in the identification of disease-specific mutations in this gene based upon comparison of normal and SMA cDNA sequence, amplification and sequencing of SMA DNA samples, and RT-PCR sequencing studies.

  12. 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.

  13. Gene recovery microdissection (GRM) a process for producing chromosome region-specific libraries of expressed genes

    SciTech Connect

    Christian, A T; Coleman, M A; Tucker, J D

    2001-02-08

    Gene Recovery Microdissection (GRM) is a unique and cost-effective process for producing chromosome region-specific libraries of expressed genes. It accelerates the pace, reduces the cost, and extends the capabilities of functional genomic research, the means by which scientists will put to life-saving, life-enhancing use their knowledge of any plant or animal genome.

  14. Optimization of radiation therapy techniques for prostate cancer with prostate-rectum spacers: a systematic review.

    PubMed

    Mok, Gary; Benz, Eileen; Vallee, Jean-Paul; Miralbell, Raymond; Zilli, Thomas

    2014-10-01

    Dose-escalated radiation therapy for localized prostate cancer improves disease control but is also associated with worse rectal toxicity. A spacer placed between the prostate and rectum can be used to displace the anterior rectal wall outside of the high-dose radiation regions and potentially minimize radiation-induced rectal toxicity. This systematic review focuses on the published data regarding the different types of commercially available prostate-rectum spacers. Dosimetric results and preliminary clinical data using prostate-rectum spacers in patients with localized prostate cancer treated by curative radiation therapy are compared and discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Optimization of Radiation Therapy Techniques for Prostate Cancer With Prostate-Rectum Spacers: A Systematic Review

    SciTech Connect

    Mok, Gary; Benz, Eileen; Vallee, Jean-Paul; Miralbell, Raymond; Zilli, Thomas

    2014-10-01

    Dose-escalated radiation therapy for localized prostate cancer improves disease control but is also associated with worse rectal toxicity. A spacer placed between the prostate and rectum can be used to displace the anterior rectal wall outside of the high-dose radiation regions and potentially minimize radiation-induced rectal toxicity. This systematic review focuses on the published data regarding the different types of commercially available prostate-rectum spacers. Dosimetric results and preliminary clinical data using prostate-rectum spacers in patients with localized prostate cancer treated by curative radiation therapy are compared and discussed.

  16. LISA telescope spacer design investigations

    NASA Astrophysics Data System (ADS)

    Sanjuan, Josep; Mueller, Guido; Livas, Jeffrey; Preston, Alix; Arsenovic, Petar; Castellucci, Kevin; Generie, Joseph; Howard, Joseph; Stebbins, Robin

    ) and materials such as Silicon Carbide (SiC) and Carbon Fiber Reinforced Plastic (CFRP) are considered to be used in the telescope spacer structure. We will describe our experimental efforts to understand and quantify the behavior of different materials and also discuss a first investigation of a specific on-axis SiC telescope spacer for LISA. This work is supported by NASA contract 00069955.

  17. Dynamic function of the alkyl spacer of acetogenins in their inhibitory action with mitochondrial complex I (NADH-ubiquinone oxidoreductase).

    PubMed

    Abe, Masato; Murai, Masatoshi; Ichimaru, Naoya; Kenmochi, Atsushi; Yoshida, Takehiko; Kubo, Akina; Kimura, Yuka; Moroda, Aki; Makabe, Hidefumi; Nishioka, Takaaki; Miyoshi, Hideto

    2005-11-15

    Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition. To elucidate the function of the alkyl spacer, two sets of systematically selected analogues were synthesized. First, the length of the spacer was varied widely. Second, the local flexibility of the spacer was specifically reduced by introducing multiple bond(s) into different regions of the spacer. The optimal length of the spacer for inhibition was approximately 13 carbon atoms. The decrease in the strength of the inhibitory effect caused by elongating the spacer from 13 carbons was much more drastic than that caused by shortening. Local flexibility in a specific region of the spacer was not important for the inhibition. These observations indicate that the active conformation of the spacer is not an extended form, and is not necessarily restricted to a certain rigid shape. Moreover, an analogue in which a spacer covering 10 carbon atoms was hardened into a rodlike shape still maintained a potent inhibitory effect. Our results strongly suggest that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion. The manner of acetogenin binding to the enzyme may not be explained by a simple "key and keyhole" analogy.

  18. Isolation and characterization of genes from the SMA candidate region

    SciTech Connect

    Thompson, T.G.; Ta, D.; Wasmuth, J.J.

    1994-09-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting alpha motor neutrons. SMA has been mapped to 5q12-13, between the genetic markers D5S435 and 38.3. The distance between these markers, which are closest on the opposite sides of the SMA locus, is {approximately}750 kb. An extensive physical map of this region has been constructed by radiation hybrid (RH) mapping and YAC contig assembly. Further analysis of the YAC contig of the SMA region revealed many deletions and duplications within the YACs. The extensive rearrangements in these YACs makes it very difficult to use them to construct a cosmid contig. The YACs and cosmids isolated thus far have been used to isolate expressed sequences using the method of exon amplification. Putative exons were then used to screen various cDNA libraries to isolate the corresponding cDNAs. Genomic sequences that cross-hybridize very strongly to many of the cDNAs are present in two different regions of chromosome 5, in the SMA region and in 5p13-14. These duplications appear to represent genes and partially processed psuedogenes in one or both of the regions and it has been difficult to determine which of the two loci is the functional gene. Other cDNAs located exclusively in the SMA region have also been found. From the two classes of cDNAs (duplicated or single copy), there are four that are good candidates for the disease gene. These include a cDNA that shows homology to a chicken neuronal specific myosin heavy chain gene, an expressed variant of promelanin concentrating hormone, k-cadherin and a glutathion-S-transferase Mu class protein. We are isolating the full length transcripts for each of these and will use them in DGGE and SSCP gel analysis of SMA patients.

  19. Discordance in variation of the ITS region and the mitochondrial COI gene in the subterranean amphipod Crangonyx islandicus.

    PubMed

    Kornobis, Etienne; Pálsson, Snæbjörn

    2011-08-01

    The amphipod Crangonyx islandicus is a recently discovered species endemic to Iceland. Populations of C. islandicus are highly structured geographically and genetically. The COI and 16S mitochondrial genes confine six monophyletic groups which have diverged for up to 5 million years within Iceland, and may present two cryptic species. To investigate the potential cryptic species status we analyse here the internal transcribed spacers (ITS1 and ITS2) and compare its variation with the patterns obtained with the mtDNA. The ITS regions present much less divergence among the geographic regions in comparison with the mtDNA, distances based on ITS1 are correlated with the COI distances as well as with geographic distances, but most of the variation is observed within individuals. The variation in the ITS region appears to have been shaped both by homogenization effect of concerted evolution and divergent evolution. A duplication of 269 base pairs is found in the ITS1 of all individuals from the southern populations, its divergence from its paralog appears to predate the split of the different groups within Iceland but some evidence point to rapid diversification after the split. This duplication does not affect the secondary structures found in the 3' and 5' ends of the sequence, suggested to have a role in the excision of the ITS1. Compensatory base changes within the ITS2 sequences which have been suggested to be a species indicator were not detected. © Springer Science+Business Media, LLC 2011

  20. 45S rDNA external transcribed spacer organization reveals new phylogenetic relationships in Avena genus.

    PubMed

    Rodrigues, Joana; Viegas, Wanda; Silva, Manuela

    2017-01-01

    The genus Avena comprises four distinct genomes organized in diploid (AA or CC), tetraploid (AABB or AACC) and hexaploid species (AACCDD), constituting an interesting model for phylogenetic analysis. The aim of this work was to characterize 45S rDNA intergenic spacer (IGS) variability in distinct species representative of Avena genome diversity-A. strigosa (AA), A. ventricosa (CvCv), A. eriantha (CpCp), A. barbata (AABB), A. murphyi (AACC), A. sativa (AACCDD) and A. sterilis (AACCDD) through the assessment of the 5' external transcribed spacer (5'-ETS), a promising IGS region for phylogenetic studies poorly studied in Avena genus. In this work, IGS length polymorphisms were detected mainly due to distinct 5'-ETS sequence types resulting from major differences in the number and organization of repeated motifs. Although species with A genome revealed a 5'-ETS organization (A-organization) similar to the one previously described in A. sativa, a distinct organization was unraveled in C genome diploid species (C-organization). Interestingly, such new organization presents a higher similarity with other Poaceae species than A-genome sequences, supporting the hypothesis of C-genome being the ancestral Avena genome. Additionally, polyploid species with both genomes mainly retain the A-genome 5'-ETS organization, confirming the preferential elimination of C-genome sequences in Avena polyploid species. Moreover, 5'-ETS sequences phylogenetic analysis consistently clustered the species studied according to ploidy and genomic constitution supporting the use of ribosomal genes to highlight Avena species evolutive pathways.

  1. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

    PubMed

    Bolotin, Alexander; Quinquis, Benoit; Sorokin, Alexei; Ehrlich, S Dusko

    2005-08-01

    Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

  2. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

    PubMed

    Bolotin, Alexander; Quinquis, Benoit; Sorokin, Alexei; Ehrlich, S Dusko

    2005-08-01

    Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

  3. The TL region gene 37 encodes a Qa-1 antigen

    PubMed Central

    1990-01-01

    Of all the biochemically defined mouse MHC class I molecules, the Qa-1 antigens are the only ones for which a gene has not been identified. Recent evidence has suggested that Qa-1 antigens are functional class I molecules and can function as restriction elements for gamma/delta T cells. We have examined the relationship between Qa-1 and the product of gene 37, a presumed novel class I antigen encoded within the TL region. Immunoprecipitation and polyacrylamide gel electrophoresis analysis of the molecules reactive with anti-Qa-1 and anti-37 sera show that the Qa-1 molecule of Qa-1b (Qa-1.2) mouse strains is identical to the product of gene 37 on the basis of molecular weight, pI, and strain distribution. Immunodepletion, biosynthetic labeling, and tunicamycin treatment confirm that the protein encoded by gene 37 in Qa-1b mice is Qa-1.2. In contrast, the anti-37 serum was unable to recognize the Qa-1 molecule in Qa-1a strains. Given the fact that the only allele to gene 37 thus far identified in a Qa-1a strain (A/J) has a termination codon in the alpha 3 domain, our data lead us to conclude that the Qa-1 molecule expressed in Qa-1a mice is not a true allelic product of the gene 37 encoded antigen of Qa-1b mouse strains. PMID:2258708

  4. Nonuniform Deposition of Pressurized Metered-Dose Aerosol in Spacer Devices.

    PubMed

    Ogrodnik, Nicholas; Azzi, Victor; Sprigge, Elizabeth; Fiset, Sandra; Matida, Edgar

    2016-12-01

    Pressurized metered-dose inhalers (pMDIs) are commonly used to administer medication to patients suffering from chronic lower respiratory tract diseases such as asthma. Inhaling medication directly from a pMDI can prove difficult for some patients and, as a result, add-on devices (or spacers) have been designed to aid in the delivery of medication. Although spacers increase the percentage of medication that reaches the patient, medication will also nonsymmetrically deposit on the walls of the device and will be lost to the device itself. The deposition of medication, salbutamol sulfate, within a large- and a small-volume spacer, has been studied through an experimental and numerical analysis. Experiments were conducted at inspiratory flow rates ranging from 30 to 60 L/min. The amount of deposition of the medication on the walls of the spacer was quantified through an application of spectrophotometry. Computational fluid dynamics was used to quantify the deposition numerically. Simulations were conducted by implementing mean flow and turbulent tracking of particles using unsteady Reynolds-averaged Navier-Stokes (URANS) equations with a shear stress transport turbulence model. Regions of deposition are of interest, as well as how the method of deposition varied for different inhalation flow rates. The deposition of salbutamol sulfate in the Volumatic(®) and OptiChamber(®) spacers was found to be greater in the lower half as opposed to the upper half of the spacer due to a downward spray angle. With an increased flow rate, these spacers demonstrated a slight increase in medication delivered to the inline filter, which is analogous to that reaching the patient, and an increase in distal deposition. For the numerical analysis, the results indicated that inertial impaction is the most likely method of deposition for the Volumatic spacer, and turbulence is more likely to cause deposition in the OptiChamber spacer.

  5. Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata.

    PubMed

    Sumida, Masayuki; Kato, Yoji; Kurabayashi, Atsushi

    2004-04-01

    The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.

  6. A Functional Analysis of the Spacer of V(D)J Recombination Signal Sequences

    PubMed Central

    Cowell, Lindsay G; Ptaszek, Leon M; Kelsoe, Garnett

    2003-01-01

    During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a “digital” requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an “analog” manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for “RSS information content.” The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein–DNA interactions in various biological systems. PMID:14551903

  7. Structure and organization of the rDNA intergenic spacer in lake trout (Salvelinus namaycush).

    PubMed

    Reed, K M; Phillips, R B

    2000-01-01

    A total-genomic cosmid library was created to isolate complete copies of the rDNA cistron of lake trout (Salvelinus namaycush) in order to study the structure and organization of the intergenic spacer (IGS) in this species. A total of 60 rDNA-positive clones (average inserts > 25 kb) was recovered by screening the library with a rDNA-specific probe. Positive clones were assayed for the presence of the two internal rDNA spacers (ITS-1 and ITS-2) and the entire IGS fragment was successfully amplified from 42 clones by PCR. Length of the IGS fragments ranged from 9.4 to 17.8 kb. Comparative restriction mapping of the IGS-PCR products of several clones indicated two regions of extensive length variation surrounding a central region with sequence conservation. DNA sequence analysis was used to investigate the molecular basis of the IGS length variation and focused on identifying the region responsible for this variation. Over 9 kb of DNA sequence was obtained for one clone (A1) with a total IGS length of approximately 12.4 kb. Sequence of a conserved central region contained two open reading frames and a number of short direct repeats. Length variation in the IGS was determined by RFLP to result from differences in the number of copies of repetitive DNA sequences. These included an 89-bp tandem repeat (alpha repeats), an 82-bp element (beta repeats), a 168-177-bp element (chi repeats), and a 179-201-bp element (delta repeats). Overall nucleotide composition of the IGS was biased towards A and T (%GC = 47.4). Maintenance of discrete rDNA-length variants in lake trout suggests that the rate of gene conversion is insufficient to produce homogeneous copies across the genome.

  8. Molecular analysis of a NOR site polymorphism in brown trout (Salmo trutta): organization of rDNA intergenic spacers.

    PubMed

    Castro, J; Sánchez, L; Martínez, P; Lucchini, S D; Nardi, I

    1997-12-01

    Using restriction endonuclease mapping, we have analyzed the organization of rDNA (DNA coding for ribosomal RNA (rRNA)) units in the salmonid fish Salmo trutta, as an initial step toward understand the molecular basis of a nucleolar organizer region (NOR) site polymorphism detected in this species. The size of the rDNA units ranged between 15 and 23 kb, with remarkable variation both within individuals and between populations. Three regions of internal tandem repetitiveness responsible for this length polymorphism were located to the intergenic spacers. NOR site polymorphic individuals showed a higher number of length classes, in some cases forming a complete 1 kb fragment ladder. The amount of rRNA genes was as much as 8-fold higher in polymorphic individuals compared with standard individuals. All individuals from the most polymorphic population showed a 14-kb insertion of unknown nature in a small proportion (below 25%) of the 28S rRNA genes.

  9. Genetic differences in internal transcribed spacer 1 between Dermanyssus gallinae from wild birds and domestic chickens.

    PubMed

    Brännström, S; Morrison, D A; Mattsson, J G; Chirico, J

    2008-06-01

    We investigated the presence of the poultry red mite or the chicken mite, Dermanyssus gallinae De Geer, Acari: Dermanyssidae, in wild bird populations in four different geographical regions of Sweden. The mites identified as D. gallinae were compared genetically with D. gallinae from egg-producing poultry farms in the same regions. The small subunit (SSU) gene, the 5.8S ribosomal RNA (rRNA) gene and the two internal transcribed spacers (ITS) of the rRNA genes were used in the genetic analysis. All D. gallinae mites had identical SSU rRNA, 5.8S rRNA and ITS2 sequences independent of their origin. By contrast, we identified significant differences in the ITS1 sequences. Based on the differences in the ITS1 sequences, the mites could be divided into two genotypes, of wild and domesticated origin, with no variation within the groups. These results imply that wild bird populations are of low importance, if any, as natural reservoirs of D. gallinae in these four geographical regions of Sweden.

  10. The Mitochondrial Genome of the Leaf-Cutter Ant Atta laevigata: A Mitogenome with a Large Number of Intergenic Spacers

    PubMed Central

    Rodovalho, Cynara de Melo; Lyra, Mariana Lúcio; Ferro, Milene; Bacci, Maurício

    2014-01-01

    In this paper we describe the nearly complete mitochondrial genome of the leaf-cutter ant Atta laevigata, assembled using transcriptomic libraries from Sanger and Illumina next generation sequencing (NGS), and PCR products. This mitogenome was found to be very large (18,729 bp), given the presence of 30 non-coding intergenic spacers (IGS) spanning 3,808 bp. A portion of the putative control region remained unsequenced. The gene content and organization correspond to that inferred for the ancestral pancrustacea, except for two tRNA gene rearrangements that have been described previously in other ants. The IGS were highly variable in length and dispersed through the mitogenome. This pattern was also found for the other hymenopterans in particular for the monophyletic Apocrita. These spacers with unknown function may be valuable for characterizing genome evolution and distinguishing closely related species and individuals. NGS provided better coverage than Sanger sequencing, especially for tRNA and ribosomal subunit genes, thus facilitating efforts to fill in sequence gaps. The results obtained showed that data from transcriptomic libraries contain valuable information for assembling mitogenomes. The present data also provide a source of molecular markers that will be very important for improving our understanding of genomic evolutionary processes and phylogenetic relationships among hymenopterans. PMID:24828084

  11. The mitochondrial genome of the leaf-cutter ant Atta laevigata: a mitogenome with a large number of intergenic spacers.

    PubMed

    Rodovalho, Cynara de Melo; Lyra, Mariana Lúcio; Ferro, Milene; Bacci, Maurício

    2014-01-01

    In this paper we describe the nearly complete mitochondrial genome of the leaf-cutter ant Atta laevigata, assembled using transcriptomic libraries from Sanger and Illumina next generation sequencing (NGS), and PCR products. This mitogenome was found to be very large (18,729 bp), given the presence of 30 non-coding intergenic spacers (IGS) spanning 3,808 bp. A portion of the putative control region remained unsequenced. The gene content and organization correspond to that inferred for the ancestral pancrustacea, except for two tRNA gene rearrangements that have been described previously in other ants. The IGS were highly variable in length and dispersed through the mitogenome. This pattern was also found for the other hymenopterans in particular for the monophyletic Apocrita. These spacers with unknown function may be valuable for characterizing genome evolution and distinguishing closely related species and individuals. NGS provided better coverage than Sanger sequencing, especially for tRNA and ribosomal subunit genes, thus facilitating efforts to fill in sequence gaps. The results obtained showed that data from transcriptomic libraries contain valuable information for assembling mitogenomes. The present data also provide a source of molecular markers that will be very important for improving our understanding of genomic evolutionary processes and phylogenetic relationships among hymenopterans.

  12. Investigation and analysis of dual-k spacer with different materials and spacer lengths for nanowire-FET performance

    NASA Astrophysics Data System (ADS)

    Ko, Hyungwoo; Kim, Jongsu; Kang, Myounggon; Shin, Hyungcheol

    2017-10-01

    In this work, dual-k spacer structures are investigated using a variety of materials along the high-k spacer length in detail. It is known that not only the higher permittivity materials of high-k spacer boost the on-current but also lower permittivity materials of low-k spacer effectively reduce the off-current. By compared the results of other various single spacers and dual-k spacers, it is HfO2/Vacuum dual-k spacer that shows relatively higher ION, ION/IOFF, better immunity of short channel effects and outstanding device performances.

  13. Genetic recombination is targeted towards gene promoter regions in dogs.

    PubMed

    Auton, Adam; Rui Li, Ying; Kidd, Jeffrey; Oliveira, Kyle; Nadel, Julie; Holloway, J Kim; Hayward, Jessica J; Cohen, Paula E; Greally, John M; Wang, Jun; Bustamante, Carlos D; Boyko, Adam R

    2013-01-01

    The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred.

  14. Genetic Recombination Is Targeted towards Gene Promoter Regions in Dogs

    PubMed Central

    Auton, Adam; Rui Li, Ying; Kidd, Jeffrey; Oliveira, Kyle; Nadel, Julie; Holloway, J. Kim; Hayward, Jessica J.; Cohen, Paula E.; Greally, John M.; Wang, Jun; Bustamante, Carlos D.; Boyko, Adam R.

    2013-01-01

    The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred. PMID:24348265

  15. Comparative evaluation of market spacer and home made spacer in the management of bronchial asthma.

    PubMed

    Rajkumar; Vatsa, H K; Gaur, S N

    2002-03-01

    A study was conducted to compare the efficacy of market available spacer (with valve) and home made spacer (without valve)--Bislery bottle. Fifteen patients of bronchial asthma were included in the study. With the use of both devices there was significant bronchodilator effect. The reversibility using Bislery bottle was same as with spacer (market available) while comparing the FVC, FEV1, FEV1/FVC %, FEF 25-75 and PEFR value. The difference in percent change in reversibility values by both the devices was not statistically significant (p < 0.05). We concluded that the Bislery bottle (without valve) is very cheap compared to market-available spacer and is equally effective which, therefore, can be substituted in bronchial asthma patients, who are unable to afford the cost of market available spacers.

  16. Defining the autism minimum candidate gene region on chromosome 7.

    PubMed

    Hutcheson, Holli B; Bradford, Y; Folstein, S E; Gardiner, M B; Santangelo, S L; Sutcliffe, J S; Haines, J L

    2003-02-01

    Previous genetic and cytogenetic studies provide evidence that points to one or more autism susceptibility genes residing on chromosome 7q (AUTS1, 115-149 cM on the Marshfield map). However, further localization using linkage analysis has proven difficult. To overcome this problem, we examined the Collaborative Linkage Study of Autism (CLSA) data-set to identify only the families potentially linked to chromosome 7. Out of 94, 47 families were identified and 17 markers were used to generate chromosomal haplotypes. We performed recombination breakpoint analysis to determine if any portion of the chromosome was predominately shared across families. The most commonly shared region spanned a 6 cM interval between D7S501 and D7S2847. Additional markers at 1 cM intervals within this region were genotyped and association and recombination breakpoint analysis was again performed. Although no significant allelic association was found, the recombination breakpoint data points to a shared region between D7S496-D7S2418 (120-123 cM) encompassing about 4.5 Mb of genomic DNA containing over 50 genes. Copyright 2003 Wiley-Liss, Inc.

  17. Characterization of Streptomyces venezuelae ATCC 10595 rRNA gene clusters and cloning of rrnA.

    PubMed Central

    La Farina, M; Stira, S; Mancuso, R; Grisanti, C

    1996-01-01

    Streptomyces venezuelae ATCC 10595 harbors seven rRNA gene clusters which can be distinguished by BglII digestion. The three rRNA genes present in each set are closely linked with the general structure 16S-23S-5S. We cloned rrnA and sequenced the 16S-23S spacer region and the region downstream of the 5S rRNA gene. No tRNA gene was found in these regions. PMID:8631730

  18. DIVERSITY OF THE TYPE 1 INTRON-ITS REGION OF THE 18S rRNA GENE IN PSEUDOGYMNOASCUS SPECIES FROM THE RED HILLS OF KANSAS.

    PubMed

    Chen, Xi; Crupper, Scott S

    2016-09-01

    Gypsum caves found throughout the Red Hills of Kansas have the state's most diverse and largest population of cave-roosting bats. White-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans, which threatens all temperate bat species, has not been previously detected in the gypsum caves as this disease moves westward from the eastern United States. Cave soil was obtained from the gypsum caves, and using the polymerase chain reaction, a 624-nucleotide DNA fragment specific to the Type 1 intron-internal transcribed spacer region of the 18S rRNA gene from Pseudogymnoascus species was amplified. Subsequent cloning and DNA sequencing indicated P. destructans DNA was present, along with 26 uncharacterized Pseudogymnoascus DNA variants. However, no evidence of WNS was observed in bat populations residing in these caves.

  19. The Effect of Spacer Morphology on the Aerosolization Performance of Metered-Dose Inhalers

    PubMed Central

    Momeni, Sepideh; Nokhodchi, Ali; Ghanbarzadeh, Saeed; Hamishehkar, Hamed

    2016-01-01

    Purpose: Respiratory drug delivery has been attracted great interest for the past decades, because of the high incidence of pulmonary diseases. However, despite its invaluable benefits, there are some major drawbacks in respiratory drug delivery, mainly due to the relatively high drug deposition in undesirable regions. One way to improve the efficiency of respiratory drug delivery through metered-dose inhalers (MDI) is placing a respiratory spacer between the inhaler exit and the mouth. The aim of this study was to assess the effect of type and shape of spacer on the aerosolization performance of MDIs. Methods: A commercial Beclomethasone Dipropionate (BDP) MDI alone or equipped with two different spacer devices (roller and pear type) widely distributed in the world pharmaceutical market was used. The effect of spacers was evaluated by calculating aerosolization indexes such as fine particle fraction (FPF), mass median aerodynamic diameters (MMAD) and geometric standard deviation (GSD) using the next generation impactor. Results: Although one of the spacers resulted in superior outcomes than the other one, but it was not statistically significant. Conclusion: The results confirmed that the type and shape of spacer did not substantially influence the aerosolization performance of MDIs. PMID:27478789

  20. Parallel evolution of genes and languages in the Caucasus region.

    PubMed

    Balanovsky, Oleg; Dibirova, Khadizhat; Dybo, Anna; Mudrak, Oleg; Frolova, Svetlana; Pocheshkhova, Elvira; Haber, Marc; Platt, Daniel; Schurr, Theodore; Haak, Wolfgang; Kuznetsova, Marina; Radzhabov, Magomed; Balaganskaya, Olga; Romanov, Alexey; Zakharova, Tatiana; Soria Hernanz, David F; Zalloua, Pierre; Koshel, Sergey; Ruhlen, Merritt; Renfrew, Colin; Wells, R Spencer; Tyler-Smith, Chris; Balanovska, Elena

    2011-10-01

    We analyzed 40 single nucleotide polymorphism and 19 short tandem repeat Y-chromosomal markers in a large sample of 1,525 indigenous individuals from 14 populations in the Caucasus and 254 additional individuals representing potential source populations. We also employed a lexicostatistical approach to reconstruct the history of the languages of the North Caucasian family spoken by the Caucasus populations. We found a different major haplogroup to be prevalent in each of four sets of populations that occupy distinct geographic regions and belong to different linguistic branches. The haplogroup frequencies correlated with geography and, even more strongly, with language. Within haplogroups, a number of haplotype clusters were shown to be specific to individual populations and languages. The data suggested a direct origin of Caucasus male lineages from the Near East, followed by high levels of isolation, differentiation, and genetic drift in situ. Comparison of genetic and linguistic reconstructions covering the last few millennia showed striking correspondences between the topology and dates of the respective gene and language trees and with documented historical events. Overall, in the Caucasus region, unmatched levels of gene-language coevolution occurred within geographically isolated populations, probably due to its mountainous terrain.

  1. Meiotic genes are enriched in regions of reduced archaic ancestry.

    PubMed

    Jégou, B; Sankararaman, S; Rolland, A D; Reich, D; Chalmel, F

    2017-04-21

    About 1-6% of the genetic ancestry of modern humans today originates from admixture with archaic humans. It has recently been shown that autosomal genomic regions with a reduced proportion of Neanderthal and Denisovan ancestries are significantly enriched in genes that are more expressed in testis than in other tissues. To determine whether a cellular segregation pattern would exist, we combined maps of archaic introgression with a cross-analysis of three transcriptomic datasets deciphering the transcriptional landscape of human gonadal cell types. We reveal that the regions deficient in both Neanderthal and Denisovan ancestries contain a significant enrichment of genes transcribed in meiotic germ cells. The interbreeding of anatomically modern humans with archaic humans may have introduced archaic-derived alleles that contributed to genetic incompatibilities affecting meiosis that were subsequently purged by natural selection. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Parallel Evolution of Genes and Languages in the Caucasus Region

    PubMed Central

    Balanovsky, Oleg; Dibirova, Khadizhat; Dybo, Anna; Mudrak, Oleg; Frolova, Svetlana; Pocheshkhova, Elvira; Haber, Marc; Platt, Daniel; Schurr, Theodore; Haak, Wolfgang; Kuznetsova, Marina; Radzhabov, Magomed; Balaganskaya, Olga; Romanov, Alexey; Zakharova, Tatiana; Soria Hernanz, David F.; Zalloua, Pierre; Koshel, Sergey; Ruhlen, Merritt; Renfrew, Colin; Wells, R. Spencer; Tyler-Smith, Chris; Balanovska, Elena

    2012-01-01

    We analyzed 40 SNP and 19 STR Y-chromosomal markers in a large sample of 1,525 indigenous individuals from 14 populations in the Caucasus and 254 additional individuals representing potential source populations. We also employed a lexicostatistical approach to reconstruct the history of the languages of the North Caucasian family spoken by the Caucasus populations. We found a different major haplogroup to be prevalent in each of four sets of populations that occupy distinct geographic regions and belong to different linguistic branches. The haplogroup frequencies correlated with geography and, even more strongly, with language. Within haplogroups, a number of haplotype clusters were shown to be specific to individual populations and languages. The data suggested a direct origin of Caucasus male lineages from the Near East, followed by high levels of isolation, differentiation and genetic drift in situ. Comparison of genetic and linguistic reconstructions covering the last few millennia showed striking correspondences between the topology and dates of the respective gene and language trees, and with documented historical events. Overall, in the Caucasus region, unmatched levels of gene-language co-evolution occurred within geographically isolated populations, probably due to its mountainous terrain. PMID:21571925

  3. Unusual structure of ribosomal DNA in the copepod Tigriopus californicus: intergenic spacer sequences lack internal subrepeats.

    PubMed

    Burton, R S; Metz, E C; Flowers, J M; Willett, C S

    2005-01-03

    Eukaryotic nuclear ribosomal DNA (rDNA) is typically arranged as a series of tandem repeats coding for 18S, 5.8S, and 28S ribosomal RNAs. Transcription of rDNA repeats is initiated in the intergenic spacer (IGS) region upstream of the 18S gene. The IGS region itself typically consists of a set of subrepeats that function as transcriptional enhancers. Two important evolutionary forces have been proposed to act on the IGS region: first, selection may favor changes in the number of subrepeats that adaptively adjust rates of rDNA transcription, and second, coevolution of IGS sequence with RNA polymerase I transcription factors may lead to species specificity of the rDNA transcription machinery. To investigate the potential role of these forces on population differentiation and hybrid breakdown in the intertidal copepod Tigriopus californicus, we have characterized the rDNA of five T. californicus populations from the Pacific Coast of North America and one sample of T. brevicornicus from Scotland. Major findings are as follows: (1) the structural genes for 18S and 28S are highly conserved across T. californicus populations, in contrast to other nuclear and mitochondrial DNA (mtDNA) genes previously studied in these populations. (2) There is extensive differentiation among populations in the IGS region; in the extreme, no homology is observed across the IGS sequences (>2 kb) from the two Tigriopus species. (3) None of the Tigriopus IGS sequences have the subrepeat structure common to other eukaryotic IGS regions. (4) Segregation of rDNA in laboratory crosses indicates that rDNA is located on at least two separate chromosomes in T. californicus. These data suggest that although IGS length polymorphism does not appear to play the adaptive role hypothesized in some other eukaryotic systems, sequence divergence in the rDNA promoter region within the IGS could lead to population specificity of transcription in hybrids.

  4. On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires

    PubMed Central

    Silas, Sukrit; Makarova, Kira S.; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R.; Fu, Becky Xu Hua; Hansen, Loren L.; Wang, David; Sullivan, Matthew B.; Millard, Andrew; Clokie, Martha R.; Bhaya, Devaki; Lambowitz, Alan M.; Kyrpides, Nikos C.

    2017-01-01

    ABSTRACT Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium—Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. PMID:28698278

  5. Anthropogenic antibiotic resistance genes mobilization to the polar regions.

    PubMed

    Hernández, Jorge; González-Acuña, Daniel

    2016-01-01

    Anthropogenic influences in the southern polar region have been rare, but lately microorganisms associated with humans have reached Antarctica, possibly from military bases, fishing boats, scientific expeditions, and/or ship-borne tourism. Studies of seawater in areas of human intervention and proximal to fresh penguin feces revealed the presence of Escherichia coli strains least resistant to antibiotics in penguins, whereas E. coli from seawater elsewhere showed resistance to one or more of the following antibiotics: ampicillin, tetracycline, streptomycin, and trim-sulfa. In seawater samples, bacteria were found carrying extended-spectrum β-lactamase (ESBL)-type CTX-M genes in which multilocus sequencing typing (MLST) showed different sequence types (STs), previously reported in humans. In the Arctic, on the contrary, people have been present for a long time, and the presence of antibiotic resistance genes (ARGs) appears to be much more wide-spread than was previously reported. Studies of E coli from Arctic birds (Bering Strait) revealed reduced susceptibility to antibiotics, but one globally spreading clone of E. coli genotype O25b-ST131, carrying genes of ESBL-type CTX-M, was identified. In the few years between sample collections in the same area, differences in resistance pattern were observed, with E. coli from birds showing resistance to a maximum of five different antibiotics. Presence of resistance-type ESBLs (TEM, SHV, and CTX-M) in E. coli and Klebsiella pneumoniae was also confirmed by specified PCR methods. MLST revealed that those bacteria carried STs that connect them to previously described strains in humans. In conclusion, bacteria previously related to humans could be found in relatively pristine environments, and presently human-associated, antibiotic-resistant bacteria have reached a high global level of distribution that they are now found even in the polar regions.

  6. Anthropogenic antibiotic resistance genes mobilization to the polar regions

    PubMed Central

    Hernández, Jorge; González-Acuña, Daniel

    2016-01-01

    Anthropogenic influences in the southern polar region have been rare, but lately microorganisms associated with humans have reached Antarctica, possibly from military bases, fishing boats, scientific expeditions, and/or ship-borne tourism. Studies of seawater in areas of human intervention and proximal to fresh penguin feces revealed the presence of Escherichia coli strains least resistant to antibiotics in penguins, whereas E. coli from seawater elsewhere showed resistance to one or more of the following antibiotics: ampicillin, tetracycline, streptomycin, and trim-sulfa. In seawater samples, bacteria were found carrying extended-spectrum β-lactamase (ESBL)-type CTX-M genes in which multilocus sequencing typing (MLST) showed different sequence types (STs), previously reported in humans. In the Arctic, on the contrary, people have been present for a long time, and the presence of antibiotic resistance genes (ARGs) appears to be much more wide-spread than was previously reported. Studies of E coli from Arctic birds (Bering Strait) revealed reduced susceptibility to antibiotics, but one globally spreading clone of E. coli genotype O25b-ST131, carrying genes of ESBL-type CTX-M, was identified. In the few years between sample collections in the same area, differences in resistance pattern were observed, with E. coli from birds showing resistance to a maximum of five different antibiotics. Presence of resistance-type ESBLs (TEM, SHV, and CTX-M) in E. coli and Klebsiella pneumoniae was also confirmed by specified PCR methods. MLST revealed that those bacteria carried STs that connect them to previously described strains in humans. In conclusion, bacteria previously related to humans could be found in relatively pristine environments, and presently human-associated, antibiotic-resistant bacteria have reached a high global level of distribution that they are now found even in the polar regions. PMID:27938628

  7. Regional genomic instability predisposes to complex dystrophin gene rearrangements.

    PubMed

    Oshima, Junko; Magner, Daniel B; Lee, Jennifer A; Breman, Amy M; Schmitt, Eric S; White, Lisa D; Crowe, Carol A; Merrill, Michelle; Jayakar, Parul; Rajadhyaksha, Aparna; Eng, Christine M; del Gaudio, Daniela

    2009-09-01

    Mutations in the dystrophin gene (DMD) cause Duchenne and Becker muscular dystrophies and the majority of cases are due to DMD gene rearrangements. Despite the high incidence of these aberrations, little is known about their causative molecular mechanism(s). We examined 792 DMD/BMD clinical samples by oligonucleotide array-CGH and report on the junction sequence analysis of 15 unique deletion cases and three complex intragenic rearrangements to elucidate potential underlying mechanism(s). Furthermore, we present three cases with intergenic rearrangements involving DMD and neighboring loci. The cases with intragenic rearrangements include an inversion with flanking deleted sequences; a duplicated segment inserted in direct orientation into a deleted region; and a splicing mutation adjacent to a deletion. Bioinformatic analysis demonstrated that 7 of 12 breakpoints combined among 3 complex cases aligned with repetitive sequences, as compared to 4 of 30 breakpoints for the 15 deletion cases. Moreover, the inversion/deletion case may involve a stem-loop structure that has contributed to the initiation of this rearrangement. For the duplication/deletion and splicing mutation/deletion cases, the presence of the first mutation, either a duplication or point mutation, may have elicited the deletion events in an attempt to correct preexisting mutations. While NHEJ is one potential mechanism for these complex rearrangements, the highly complex junction sequence of the inversion/deletion case suggests the involvement of a replication-based mechanism. Our results support the notion that regional genomic instability, aided by the presence of repetitive elements, a stem-loop structure, and possibly preexisting mutations, may elicit complex rearrangements of the DMD gene.

  8. Phylogeography and conservation genetics of Hygrophila pogonocalyx (Acanthaceae) based on atpB-rbcL noncoding spacer cpDNA.

    PubMed

    Huang, Jao-Ching; Wang, Wei-Kuang; Peng, Ching-I; Chiang, Tzen-Yuh

    2005-02-01

    Genetic variation in the atpB-rbcL intergenic spacer region of chloroplast DNA (cpDNA) was investigated in Hygrophila pogonocalyx Hayata (Acanthaceae), an endangered and endemic species in Taiwan. In this aquatic species, seed dispersal from capsules via elasticity is constrained by gravity and is thereby confined within populations, resulting in limited gene flow between populations. In this study, a total of 849 bp of the cpDNA atpB-rbcL spacer were sequenced from eight populations of H. pogonocalyx. Nucleotide diversity in the cpDNA is low (theta = 0.00343+/-0.00041). The distribution of genetic variation among populations agrees with an "isolation-by-distance" model. Two geographically correlated groups, the western and eastern regions, were identified in a neighbor-joining tree and a minimum-spanning network. Phylogeographical analyses based on the cpDNA network suggest that the present-day differentiation between western and eastern groups of H. pogonocalyx resulted from past fragmentation. The differentiation between eastern and western populations may be ascribed to isolation since the formation of the Central Mountain Range about 5 million years ago, which is consistent with the rate estimates based on a molecular clock of cpDNA.

  9. Separator-spacer for electrochemical systems

    DOEpatents

    Grimes, Patrick G.; Einstein, Harry; Newby, Kenneth R.; Bellows, Richard J.

    1983-08-02

    An electrochemical cell construction features a novel co-extruded plastic electrode in an interleaved construction with a novel integral separator-spacer. Also featured is a leak and impact resistant construction for preventing the spill of corrosive materials in the event of rupture.

  10. Funhaler spacer: improving adherence without compromising delivery

    PubMed Central

    Watt, P; Clements, B; Devadason, S; Chaney, G

    2003-01-01

    A novel asthma spacer device, the "Funhaler", incorporates incentive toys which are isolated from the main inspiratory circuit by a valve. Here we show that its use does not compromise drug delivery. Improved adherence combined with satisfactory delivery characteristics suggest that the Funhaler may be useful for management of young asthmatics. PMID:12818901

  11. Detection of multiple infections by Monocystis strains in a single earthworm host using ribosomal internal transcribed spacer sequence variation.

    PubMed

    Velavan, T P; Schulenburg, H; Michiels, N K

    2010-01-01

    Monocystis sp. are sporocyst-forming apicomplexan parasites common in seminal vesicles of the earthworm Lumbricus terrestris where they may account for temporary castration. This study describes the internal transcribed spacer (ITS) region of the ribosomal cistron of Monocystis sp. This region, including ITS-1, the 5.8S ribosomal RNA gene, and ITS-2, was PCR amplified, cloned, and sequenced for Monocystis sp. isolated from the seminal vesicles of several wild-caught L. terrestris. Our analysis revealed substantial polymorphisms, also within single host organisms, indicating intra-host diversity of parasites. These genetic markers are the first that allow distinction of Monocystis sp. genotypes, opening new avenues for the study of parasite diversity within and between hosts.

  12. Mutations in BTD gene causing biotinidase deficiency: a regional report.

    PubMed

    Kasapkara, Çiğdem Seher; Akar, Melek; Özbek, Mehmet Nuri; Tüzün, Heybet; Aldudak, Bedri; Baran, Rıza Taner; Tanyalçın, Tijen

    2015-03-01

    Biotinidase deficiency is an autosomal recessive inborn error of biotin metabolism. Children with biotinidase deficiency cannot cleave biocytin and, therefore, cannot recycle biotin. Untreated individuals become secondarily biotin deficient, which in turn results in decreased activities of the biotin-dependent carboxylases and the subsequent accumulation of toxic metabolites causing clinical symptoms. Biotinidase deficiency is characterized by neurological, cutaneous manifestations and metabolic abnormalities. The worldwide incidence of profound biotinidase deficiency has been estimated at 1:112,271. The human biotinidase gene is located on chromosome 3p25 and consists of four exons with a total length of 1629 base pairs. To date, more than 100 mutations in the biotinidase gene known to cause biotinidase deficiency have been reported. The vast majority of mutations are homozygous or compound heterozygous. Finding known mutations can be correlated with the biochemical enzymatic results. This report summarizes the demographic features of patients identified as biotinidase deficient from August of 2012 through August of 2013 and mutation analysis results for 20 cases in the southeast region of Turkey.

  13. The low-recombining pericentromeric region of barley restricts gene diversity and evolution but not gene expression

    PubMed Central

    Baker, Katie; Bayer, Micha; Cook, Nicola; Dreißig, Steven; Dhillon, Taniya; Russell, Joanne; Hedley, Pete E; Morris, Jenny; Ramsay, Luke; Colas, Isabelle; Waugh, Robbie; Steffenson, Brian; Milne, Iain; Stephen, Gordon; Marshall, David; Flavell, Andrew J

    2014-01-01

    The low-recombining pericentromeric region of the barley genome contains roughly a quarter of the genes of the species, embedded in low-recombining DNA that is rich in repeats and repressive chromatin signatures. We have investigated the effects of pericentromeric region residency upon the expression, diversity and evolution of these genes. We observe no significant difference in average transcript level or developmental RNA specificity between the barley pericentromeric region and the rest of the genome. In contrast, all of the evolutionary parameters studied here show evidence of compromised gene evolution in this region. First, genes within the pericentromeric region of wild barley show reduced diversity and significantly weakened purifying selection compared with the rest of the genome. Second, gene duplicates (ohnolog pairs) derived from the cereal whole-genome duplication event ca. 60MYa have been completely eliminated from the barley pericentromeric region. Third, local gene duplication in the pericentromeric region is reduced by 29% relative to the rest of the genome. Thus, the pericentromeric region of barley is a permissive environment for gene expression but has restricted gene evolution in a sizeable fraction of barley's genes. PMID:24947331

  14. Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars.

    PubMed Central

    Laguerre, G; Mavingui, P; Allard, M R; Charnay, M P; Louvrier, P; Mazurier, S I; Rigottier-Gois, L; Amarger, N

    1996-01-01

    Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient. PMID:8787401

  15. Suitability of internal transcribed spacers (ITS) as markers for the population genetic structure of Blastocystis spp.

    PubMed

    Villalobos, Guiehdani; Orozco-Mosqueda, Guadalupe Erendira; Lopez-Perez, Merle; Lopez-Escamilla, Eduardo; Córdoba-Aguilar, Alex; Rangel-Gamboa, Lucia; Olivo-Diaz, Angelica; Romero-Valdovinos, Mirza; Maravilla, Pablo; Martinez-Hernandez, Fernando

    2014-10-03

    The purpose of this study was to assess the genetic variation and differentiation of Blastocystis subtypes (STs) recovered from symptomatic children by analysing partial sequences of the small subunit rDNA gene region (SSUrDNA) and internal transcribed spacers (1 and 2) plus the 5.8S region (ITS, ITS1 + 5.8S + ITS2) and comparing with isolates from other countries. Faecal samples from 47 Blastocystis-infected children with gastrointestinal symptoms and negative for pathogenic enterobacteria were analysed. PCR was performed on DNA from all the samples to identify Blastocystis STs, amplifying a fragment of SSUrDNA and the ITS region. The amplicons were purified and sequenced, and consensus sequences were submitted to GenBank; afterwards, SSUrDNA sequences were analysed for genetic diversity according to geographic area. Regarding the Blastocystis STs found, 51% were ST1, 23% ST2, 19% ST3 and 2% ST7. For ITS, a haplotype network tree and Bayesian inference revealed the presence of two novel variants of ST1, clustering some sequences into ST1A and ST1B. The values of nucleotide diversity (π) and haplotype polymorphism (θ) for ST1, ST2 and ST3 ranged from 0 to 1, whereas the ratio of genetic differentiation (FST)/migration index (Nm) showed the highest differentiation between Libya and Thailand-Philippines for ST2 (0.282/0.63). In contrast, a high flow gene was observed between Czech Republic-Denmark-Holland-Spain and USA-Mexico-Colombia for ST1 (0.003/84). Our data on genetic differentiation and gene flow might explain the differences for the prevalence of Blastocystis STs. Moreover, the ITS region could be used as a genetic marker to assess genetic variation in this parasite.

  16. Identification and characterization of a juvenile hormone (JH) response region in the JH esterase gene from the spruce budworm, Choristoneura fumiferana.

    PubMed

    Kethidi, Damodar R; Perera, Srini C; Zheng, S; Feng, Qi-Li; Krell, Peter; Retnakaran, Arthur; Palli, Subba R

    2004-05-07

    Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and 20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.

  17. [Effect of spacer on red and green phosphorescent organic light-emitting devices].

    PubMed

    Zhang, Wei; Zhang, Fang-Hui; Huang, Jin

    2014-02-01

    We have investigated the performances of organic light-emitting diodes (OLED) with different spacer, the structure was fabricated as ITO/MoO3 (40 nm)/NPB(40 nm)/TCTA(10 nm)/CBP:GIr114:R-4B2%(30 nm)/spacer (3 nm)/CBP: GIr114%:R-4B2%(30 nm)/BCP(10 nm)/Alq3 (40 nm)/LiF(1 nm)/Al(100 nm), the spacers were CBP, TCTA, TPBI and BCP separately, GIr1 and R-4B were green and red phosphorescent dye respectively. The results showed that compared to the reference device utilized CBP as the spacer layer, TCTA, TPBI and BCP had higher current efficiency in excess of 59%, 79% and 93%, the maximum current efficiency of 16.91 cd x A(-1) was achieved with BCP as the spacer at voltage of 5 V, TPBI and BCP as the spacer layer obtained the higher current density and lower efficiency roll-off. We attributed to these results to the follow reasons, the first was that carriers and excitons were limited to a narrow recombination region because of TCTA with higher LUMO energy level and triplet energy, which improved the probability of carriers recombination, in addition, more serious quenching at higher current density. The second reason was that TPBI and BCP had the higher HOMO energy level and electron mobility, which broadened excitons recombination zone. In addition, the spacer layer caused the accumulation of electrons or holes and the formation of high space electric field, leading to carrier injection and transport more effectively. In particular, we obtained a better stability of phosphorescent organic light-emitting diodes since the way for the red and green co-doped with host material.

  18. 45S rDNA external transcribed spacer organization reveals new phylogenetic relationships in Avena genus

    PubMed Central

    Rodrigues, Joana; Viegas, Wanda

    2017-01-01

    The genus Avena comprises four distinct genomes organized in diploid (AA or CC), tetraploid (AABB or AACC) and hexaploid species (AACCDD), constituting an interesting model for phylogenetic analysis. The aim of this work was to characterize 45S rDNA intergenic spacer (IGS) variability in distinct species representative of Avena genome diversity–A. strigosa (AA), A. ventricosa (CvCv), A. eriantha (CpCp), A. barbata (AABB), A. murphyi (AACC), A. sativa (AACCDD) and A. sterilis (AACCDD) through the assessment of the 5’ external transcribed spacer (5’-ETS), a promising IGS region for phylogenetic studies poorly studied in Avena genus. In this work, IGS length polymorphisms were detected mainly due to distinct 5’-ETS sequence types resulting from major differences in the number and organization of repeated motifs. Although species with A genome revealed a 5’-ETS organization (A-organization) similar to the one previously described in A. sativa, a distinct organization was unraveled in C genome diploid species (C-organization). Interestingly, such new organization presents a higher similarity with other Poaceae species than A-genome sequences, supporting the hypothesis of C-genome being the ancestral Avena genome. Additionally, polyploid species with both genomes mainly retain the A-genome 5’-ETS organization, confirming the preferential elimination of C-genome sequences in Avena polyploid species. Moreover, 5’-ETS sequences phylogenetic analysis consistently clustered the species studied according to ploidy and genomic constitution supporting the use of ribosomal genes to highlight Avena species evolutive pathways. PMID:28448637

  19. T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis.

    PubMed

    Cai, Chen-Leng; Zhou, Wenlai; Yang, Lei; Bu, Lei; Qyang, Yibing; Zhang, Xiaoxue; Li, Xiaodong; Rosenfeld, Michael G; Chen, Ju; Evans, Sylvia

    2005-05-01

    Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. We provide evidence that Tbx2 is a direct target for repression by Tbx20 in developing heart. We have also found that Tbx2 directly binds to the Nmyc1 promoter in developing heart, and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. Our data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. We present a model whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, we have found that Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development.

  20. Updates on quick identification of acetic acid bacteria with a focus on the 16S-23S rRNA gene internal transcribed spacer and the analysis of cell proteins by MALDI-TOF mass spectrometry.

    PubMed

    Trček, Janja; Barja, François

    2015-03-02

    Acetic acid bacteria have attracted much attention over the past few years, due mainly to their metabolic traits that are of interest to the biotechnology industry. In addition, it turns out that their ecological habitats are almost unlimited since they have been found as symbionts in different insects and also as emerging opportunistic human pathogens. Very surprising is the finding that they colonize niches considered anaerobic, disproving the generalized statement that they are strict aerobes. Since they have taken on different biological roles in our environment, more and more people are charged with the task of identifying them. However, this turns out to be not always easy, especially if we are using phenotypic approaches for identification. A substantial step forward in making the identification of acetic acid bacteria easier was made possible using molecular biological methods, which have been extensively tested since 2000. However, some molecular methods require expensive machines and experienced staff, and moreover the level of their discrimination varies. All these factors must be considered when selecting the most appropriate approach for identifying acetic acid bacteria. With this objective in mind, this review article discusses the benefits and drawbacks of molecular biological methods for identification of acetic acid bacteria, with a focus on the 16S-23S rRNA gene ITS regions and the recently described alternative method for identification of acetic acid bacteria, MALDI-TOF MS.

  1. Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer

    PubMed Central

    Ruegger, Paul M.; Clark, Robin T.; Weger, John R.; Braun, Jonathan; Borneman, James

    2014-01-01

    Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses. PMID:25034229

  2. Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer.

    PubMed

    Ruegger, Paul M; Clark, Robin T; Weger, John R; Braun, Jonathan; Borneman, James

    2014-10-01

    Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses.

  3. Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression.

    PubMed Central

    Lloyd, J A; Lee, R F; Lingrel, J B

    1989-01-01

    Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator. Images PMID:2472607

  4. Tube support grid and spacer therefor

    DOEpatents

    Ringsmuth, Richard J.; Kaufman, Jay S.

    1986-01-01

    A tube support grid and spacers therefor provide radially inward preloading of heat exchange tubes to minimize stress upon base welds due to differential thermal expansion. The grid comprises a concentric series of rings and spacers with opposing concave sides for conforming to the tubes and V-shaped ends to provide resilient flexibility. The flexibility aids in assembly and in transmitting seismic vibrations from the tubes to a shroud. The tube support grid may be assembled in place to achieve the desired inwardly radial preloading of the heat exchange tubes. Tab and slot assembly further minimizes stresses in the system. The radii of the grid rings may be preselected to effect the desired radially inward preloading.

  5. Heterogeneous diversity of spacers within CRISPR

    NASA Astrophysics Data System (ADS)

    Deem, Michael; He, Jiankui

    2011-03-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) in bacterial and archaeal DNA have recently been shown to be a new type of anti-viral immune system in these organisms. We here study the diversity of spacers in CRISPR under selective pressure. We propose a population dynamics model that explains the biological observation that the leader-proximal end of CRISPR is more diversified and the leader-distal end of CRISPR is more conserved. This result is shown to be in agreement with recent experiments. Our results show that the CRISPR spacer structure is influenced by and provides a record of the viral challenges that bacteria face. 1) J. He and M. W. Deem, Phys. Rev. Lett. 105 (2010) 128102

  6. Improved nuclear fuel assembly grid spacer

    DOEpatents

    Marshall, John; Kaplan, Samuel

    1977-01-01

    An improved fuel assembly grid spacer and method of retaining the basic fuel rod support elements in position within the fuel assembly containment channel. The improvement involves attachment of the grids to the hexagonal channel and of forming the basic fuel rod support element into a grid structure, which provides a design which is insensitive to potential channel distortion (ballooning) at high fluence levels. In addition the improved method eliminates problems associated with component fabrication and assembly.

  7. Investigation of some commercially available spacer devices for the delivery of glucocorticoid steroids from a pMDI.

    PubMed

    Williams, R O; Patel, A M; Barron, M K; Rogers, T L

    2001-05-01

    were used (P < .05). The GSD was not influenced by the use of a spacer device. The use of a spacer device will enhance pMDI therapy by reducing the amount of drug deposited in the oropharyngeal region, which will lead to fewer instances of local and systemic side effects. In addition, the spacer devices investigated will allow a higher dose of drug to reach the deep lung, which may permit the use of lower dosage regimens with increased therapeutic efficacy.

  8. Effects of spacer orientations on the cake formation during membrane fouling: Quantitative analysis based on 3D OCT imaging.

    PubMed

    Liu, Xin; Li, Weiyi; Chong, Tzyy Haur; Fane, Anthony G

    2017-03-01

    Spacer design plays an important role in improving the performance of membrane processes for water/wastewater treatment. This work focused on a fundamental issue of spacer design, i.e., investigating the effects of spacer orientations on the fouling behavior during a membrane process. A series of fouling experiments with different spacer orientation were carried out to in situ characterize the formation of a cake layer in a spacer unit cell via 3D optical coherence tomography (OCT) imaging. The cake layers formed at different times were digitalized for quantitatively analyzing the variation in the cake morphology as a function of time. In particular, the local deposition rates were evaluated to determine the active regions where the instantaneous changes in deposit thickness were significant. The characterization results indicate that varying the spacer orientation could substantially change the evolution of membrane fouling by particulate foulants and thereby result in a cake layer with various morphologies; the competition between growth and erosion at different locations would instantaneously respond to the micro-hydrodynamic environment that might change with time. This work confirms that the OCT-based characterization method is a powerful tool for exploring novel spacer design.

  9. Self-aligned quadruple patterning using spacer on spacer integration optimization for N5

    NASA Astrophysics Data System (ADS)

    Thibaut, Sophie; Raley, Angélique; Mohanty, Nihar; Kal, Subhadeep; Liu, Eric; Ko, Akiteru; O'Meara, David; Tapily, Kandabara; Biolsi, Peter

    2017-04-01

    To meet scaling requirements, the semiconductor industry has extended 193nm immersion lithography beyond its minimum pitch limitation using multiple patterning schemes such as self-aligned double patterning, self-aligned quadruple patterning and litho-etch / litho etch iterations. Those techniques have been declined in numerous options in the last few years. Spacer on spacer pitch splitting integration has been proven to show multiple advantages compared to conventional pitch splitting approach. Reducing the number of pattern transfer steps associated with sacrificial layers resulted in significant decrease of cost and an overall simplification of the double pitch split technique. While demonstrating attractive aspects, SAQP spacer on spacer flow brings challenges of its own. Namely, material set selections and etch chemistry development for adequate selectivities, mandrel shape and spacer shape engineering to improve edge placement error (EPE). In this paper we follow up and extend upon our previous learning and proceed into more details on the robustness of the integration in regards to final pattern transfer and full wafer critical dimension uniformity. Furthermore, since the number of intermediate steps is reduced, one will expect improved uniformity and pitch walking control. This assertion will be verified through a thorough pitch walking analysis.

  10. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Lee, Amy S Y; Engelman, Alan; Doudna, Jennifer A

    2015-03-12

    Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

  11. Heat transfer near spacer grids in rod bundles

    SciTech Connect

    Yoder, G.L.

    1985-01-01

    Heat transfer data from several sources have been assembled which show the effect of spacer grids on local heat transfer within a rod bundle. Both single phase (air and steam) data and two phase (steam/water) data show heat transfer augmentation in the grid region. Heat transfer improvement immediately beyond the grid ranges from a few percent to over fifty percent in these experiments, depending on flow conditions. The data are examined using several nondimensional parameters which relate the above effects to known quantities. The relative effect of the grid on local heat transfer is altered by both the Reynolds number and blockage ratio. Twenty to thirty hydraulic diameters are required before the local effect of the grid dissipates. Locally, both the single phase and two phase data show the same trends. Comparison of the single and two phase data also shown some differences. Some film boiling data indicate that an altered heat transfer regime may exist near the grid. High rod heat transfer coefficients at the grid locations indicate either a rewet of the rods or at least a change from film boiling to transition boiling near the spacer. The comparison also indicates that the film boiling data is affected on a global as well as local basis. This is due to the effect of the grid on the liquid distribution.

  12. Functions of milk protein gene 5' flanking regions on human growth hormone gene.

    PubMed

    Ninomiya, T; Hirabayashi, M; Sagara, J; Yuki, A

    1994-03-01

    Fragments containing 5' flanking regions of four bovine milk protein genes--alpha lactalbumin (b alpha LA), alpha S1 casein (b alpha S1CN), beta casein (b beta CN), kappa casein (b kappa CN)--and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5' regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying b alpha LA/, b beta CN/, and mWAP/hGH, and 33% of the lactated b alpha S1CN/hGH rats secreted detectable amounts of hGH (> 0.05 microgram/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of b alpha LA/, b beta CN/, b alpha S1CN/, and mWAP/hGH rats were 1.13-4,360 micrograms/ml, 0.11-10,900 micrograms/ml, 86.8-6,480 micrograms/ml, and 6.87-151 micrograms/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 microgram/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans.

    PubMed

    Nakano, Tadao; Okamoto, Munehiro; Ikeda, Yatsukaho; Hasegawa, Hideo

    2006-12-01

    Sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, nuclear internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA), and 5S rDNA of Enterobius vermicularis from captive chimpanzees in five zoos/institutions in Japan were analyzed and compared with those of pinworm eggs from humans in Japan. Three major types of variants appearing in both CO1 and ITS2 sequences, but showing no apparent connection, were observed among materials collected from the chimpanzees. Each one of them was also observed in pinworms in humans. Sequences of 5S rDNA were identical in the materials from chimpanzees and humans. Phylogenetic analysis of CO1 gene revealed three clusters with high bootstrap value, suggesting considerable divergence, presumably correlated with human evolution, has occurred in the human pinworms. The synonymy of E. gregorii with E. vermicularis is supported by the molecular evidence.

  14. Sequence analysis of the ribosomal internal transcribed spacer DNA of the crayfish parasite Psorospermium haeckeli.

    PubMed

    Bangyeekhun, E; Ryynänen, H J; Henttonen, P; Huner, J V; Cerenius, L; Söderhäll, K

    2001-10-08

    Two morphotypes of the crayfish parasite Psorospermium haeckeli were isolated from 2 crayfish species of different geographical origin. The oval-shaped sporocysts were obtained from the epidermal and connective tissue beneath the carapace of the noble crayfish Astacus astacus from Sweden and Finland. Elongated spores were isolated from the abdominal muscle tissue of the red swamp crayfish Procambarus clarkii from USA. To compare genetic divergence of 2 morphotypes of the parasite, the ribosomal internal transcribed spacer (ITS) DNA (ITS 1 and ITS 2) and the 5.8S rRNA gene were cloned and sequenced. The analysed region is variable in length, with the ribosomal ITS sequence of the European morphotype longer than the North American one. Sequence diversity is found mainly in ITS 1 and ITS 2 regions, and there is 66% and 58% similarity between the 2 morphotypes, respectively. Thus, analysis of the ribosomal ITS DNA suggests that P. haeckeli forms obtained from Europe and North America are genetically diverse, which supports the previously reported morphological characteristics.

  15. Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products.

    PubMed

    Buehler, A J; Evanowski, R L; Martin, N H; Boor, K J; Wiedmann, M

    2017-09-13

    Fungi are important spoilage organisms in dairy products. However, little is known about the diversity of naturally occurring spoilage fungi in raw milk and processed dairy products, due at least in part to the fact that classical fungal identification methods require considerable expertise. To gain further insight into the fungal diversity in the dairy system, we isolated fungi from raw milk, raw and pasteurized milk cheese, and yogurt using the selective dichloran rose bengal chloramphenicol agar. In total, 361 fungal isolates were obtained and further characterized by DNA sequencing of the internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) rRNA gene if needed. We conducted BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the ITS region sequences against the UNITE Database (https://unite.ut.ee/analysis.php), and selected other databases if needed, which allowed identification to the species level of 183 isolates and to the genus level of 107 of the 346 isolates that were successfully ITS sequenced. The isolates characterized represented 3 phyla and 19 genera; the most common genera isolated were Penicillium (25% of isolates), Debaryomyces (18%), and Candida (9%). This study not only provides, by using modern molecular tools, a baseline understanding of the types of fungi in dairy products, but also confirms that ITS sequencing is a useful approach for identification of fungal organisms found in the dairy food chain. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. Formation of plant metabolic gene clusters within dynamic chromosomal regions

    PubMed Central

    Field, Ben; Fiston-Lavier, Anna-Sophie; Kemen, Ariane; Geisler, Katrin; Quesneville, Hadi; Osbourn, Anne E.

    2011-01-01

    In bacteria, genes with related functions often are grouped together in operons and are cotranscribed as a single polycistronic mRNA. In eukaryotes, functionally related genes generally are scattered across the genome. Notable exceptions include gene clusters for catabolic pathways in yeast, synthesis of secondary metabolites in filamentous fungi, and the major histocompatibility complex in animals. Until quite recently it was thought that gene clusters in plants were restricted to tandem duplicates (for example, arrays of leucine-rich repeat disease-resistance genes). However, operon-like clusters of coregulated nonhomologous genes are an emerging theme in plant biology, where they may be involved in the synthesis of certain defense compounds. These clusters are unlikely to have arisen by horizontal gene transfer, and the mechanisms behind their formation are poorly understood. Previously in thale cress (Arabidopsis thaliana) we identified an operon-like gene cluster that is required for the synthesis and modification of the triterpene thalianol. Here we characterize a second operon-like triterpene cluster (the marneral cluster) from A. thaliana, compare the features of these two clusters, and investigate the evolutionary events that have led to cluster formation. We conclude that common mechanisms are likely to underlie the assembly and control of operon-like gene clusters in plants. PMID:21876149

  17. Differentiation of Debaryomyces hansenii and Candida famata by rRNA gene intergenic spacer fingerprinting and reassessment of phylogenetic relationships among D. hansenii, C. famata, D. fabryi, C. flareri (=D. subglobosus) and D. prosopidis: description of D. vietnamensis sp. nov. closely related to D. nepalensis.

    PubMed

    Nguyen, Huu-Vang; Gaillardin, Claude; Neuvéglise, Cécile

    2009-06-01

    The intergenic spacer rDNA amplification and AluI fingerprinting (IGSAF) method detected four distinct groups among 170 Debaryomyces hansenii strains: D. hansenii var. hansenii; Candida famata var. famata; D. hansenii var. fabryi and C. famata var. flareri. IGS sequence comparison of representative strains showed that D. hansenii var. hansenii and C. famata var. famata belonged to one species, whereas D. hansenii var. fabryi and C. famata var. flareri belonged to two different ones. This confirmed the following three species recently reinstated: D. hansenii (=C. famata), Debaryomyces fabryi and Debaryomyces subglobosus (=Candida flareri). Accordingly, growth at 37 degrees C may no longer be used to differentiate D. hansenii from D. fabryi. Riboflavin production is more specific for D. fabryi and D. subglobosus strains. IGSAF identified all the other 17 species of the genus Debaryomyces, six of them sharing with D. hansenii an rRNA gene unit harbouring two 5S rRNA genes. The phylogenetic tree established with IGS sequences was congruent with the one based on ACT1, GPD1 and COX2 sequences depicting a distinct D. hansenii clade close to the D. subglobosus, Debaryomyces prosopidis and D. fabryi clade. Description of Debaryomyces vietnamensis sp. nov. (type strain CBS 10535(T), MUCL 51648(T)), closely related to Debaryomyces nepalensis is given.

  18. NEUTRONIC REACTOR SHIELD AND SPACER CONSTRUCTION

    DOEpatents

    Wigner, E.P.; Ohlinger, L.A.

    1958-11-18

    Reactors of the heterogeneous, graphite moderated, fluid cooled type and shielding and spacing plugs for the coolant channels thereof are reported. In this design, the coolant passages extend horizontally through the moderator structure, accommodating the fuel elements in abutting end-to-end relationship, and have access openings through the outer shield at one face of the reactor to facilitate loading of the fuel elements. In the outer ends of the channels which extend through the shields are provided spacers and shielding plugs designed to offer minimal reslstance to coolant fluid flow while preventing emanation of harmful radiation through the access openings when closed between loadings.

  19. Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe.

    PubMed Central

    Lalev, A I; Nazar, R N

    1999-01-01

    The structure of the internal transcribed spacer 2 (ITS2) in Schizosaccharomyces pombe was re-evaluated with respect to phylogenetically conserved features in yeasts, features in other transcribed spacer regions as well as the binding of transacting factors which potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a very simple core structure consisting of a single extended hairpin which includes the interacting termini of the mature 5.8S and 25S rRNAs. Comparisons with ITS2 sequences in greatly diverging organisms indicate that the same feature also can be recognized. This is especially clear in organisms that contain very short sequences in which the putative structures are much less ambiguous. Diversity between organisms is the result of changes in hairpin length as well as the addition of branched helices. Protein binding and gel retardation studies with the S.pombe ITS2 further indicate that, as observed in the 3" external transcribed spacer (ETS) and ITS1 regions, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data suggest that the spacer regions all may act in a similar fashion, not only to organize the maturing terminal sequences, but also serve to organize specific soluble factors possibly acting with snoRNAs or in a manner which is analogous with that of the free snoRNPs. PMID:10454602

  20. Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

    PubMed

    Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

    2015-08-03

    Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

  1. Gene-Based Analysis of Regionally Enriched Cortical Genes in GWAS Data Sets of Cognitive Traits and Psychiatric Disorders

    PubMed Central

    Ersland, Kari M.; Christoforou, Andrea; Stansberg, Christine; Espeseth, Thomas; Mattheisen, Manuel; Mattingsdal, Morten; Hardarson, Gudmundur A.; Hansen, Thomas; Fernandes, Carla P. D.; Giddaluru, Sudheer; Breuer, René; Strohmaier, Jana; Djurovic, Srdjan; Nöthen, Markus M.; Rietschel, Marcella; Lundervold, Astri J.; Werge, Thomas; Cichon, Sven; Andreassen, Ole A.; Reinvang, Ivar; Steen, Vidar M.; Le Hellard, Stephanie

    2012-01-01

    Background Despite its estimated high heritability, the genetic architecture leading to differences in cognitive performance remains poorly understood. Different cortical regions play important roles in normal cognitive functioning and impairment. Recently, we reported on sets of regionally enriched genes in three different cortical areas (frontomedial, temporal and occipital cortices) of the adult rat brain. It has been suggested that genes preferentially, or specifically, expressed in one region or organ reflect functional specialisation. Employing a gene-based approach to the analysis, we used the regionally enriched cortical genes to mine a genome-wide association study (GWAS) of the Norwegian Cognitive NeuroGenetics (NCNG) sample of healthy adults for association to nine psychometric tests measures. In addition, we explored GWAS data sets for the serious psychiatric disorders schizophrenia (SCZ) (n = 3 samples) and bipolar affective disorder (BP) (n = 3 samples), to which cognitive impairment is linked. Principal Findings At the single gene level, the temporal cortex enriched gene RAR-related orphan receptor B (RORB) showed the strongest overall association, namely to a test of verbal intelligence (Vocabulary, P = 7.7E-04). We also applied gene set enrichment analysis (GSEA) to test the candidate genes, as gene sets, for enrichment of association signal in the NCNG GWAS and in GWASs of BP and of SCZ. We found that genes differentially expressed in the temporal cortex showed a significant enrichment of association signal in a test measure of non-verbal intelligence (Reasoning) in the NCNG sample. Conclusion Our gene-based approach suggests that RORB could be involved in verbal intelligence differences, while the genes enriched in the temporal cortex might be important to intellectual functions as measured by a test of reasoning in the healthy population. These findings warrant further replication in independent samples on cognitive traits. PMID

  2. Polymorphism in transmembrane region of MICA gene and cholelithiasis

    PubMed Central

    Shih, Shou-Chuan; Lee, Yann-Jinn; Liu, Hsin-Fu; Dang, Ching-Wen; Chang, Shih-Chuan; Lin, Shee-Chan; Kao, Chin-Roa

    2003-01-01

    AIM: To study the significance of polymorphism of MHC class I chain-related gene A (MICA) gene in patients with cholelithiasis. METHODS: Subjects included 170 unrelated adults (83 males) with cholelithiasis and 245 randomly selected unrelated adults (130 males) as controls. DNA was extracted from peripheral leukocytes and analyzed for polymorphism of 5 alleles (A4, A5, A5.1, A6 and A9) of the MICA gene. RESULTS: There was no significant difference in phenotype, allele, and genotype frequencies of any of the 5 alleles between cholelithiasis patients and controls. CONCLUSION: This study demonstrates that MICA alleles studied bear no relation to cholelithiasis. PMID:12854159

  3. The spacer arm length in cell-penetrating peptides influences chitosan/siRNA nanoparticle delivery for pulmonary inflammation treatment

    NASA Astrophysics Data System (ADS)

    Jeong, Eun Ju; Choi, Moonhwan; Lee, Jangwook; Rhim, Taiyoun; Lee, Kuen Yong

    2015-11-01

    Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of R9Gn-chitosan/siRNA nanoparticles were investigated in vitro. Increasing the spacing arm length did not significantly affect the complex formation between R9Gn-chitosan and siRNA. However, R9G10-chitosan was much more effective in delivering genes both in vitro and in vivo compared with non-modified chitosan (without the peptide) and R9-chitosan (without the spacer arm). Chitosan derivatives modified with oligoarginine containing a spacer arm can be considered as potential delivery vehicles for various genes.Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of

  4. Properties of cellulase immobilized on agarose gel with spacer

    SciTech Connect

    Chim-anage, P.; Kashiwagi, Y.; Magae, Y.; Ohta, T.; Sasaki, T.

    1986-12-01

    Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated Ch-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose. 10 references.

  5. The development process for a new spacer device.

    PubMed

    Watson, Paul

    The British Thoracic Society and Scottish Intercollegiate Guidelines Network recommend that children up to the age of five should use a pressurised metered dose inhaler with a spacer device to deliver inhaled steroids. However, large-volume spacers can be cumbersome, which is why I designed a smaller, more portable device to encourage spacer use. After prototypes were made, the idea was presented to the local NHS innovations department. With its advice and assistance, a collapsible spacer device has been developed. This article describes the product development process.

  6. Integrative Gene Regulatory Network Analysis Reveals Light-Induced Regional Gene Expression Phase Shift Programs in the Mouse Suprachiasmatic Nucleus

    PubMed Central

    Zhu, Haisun; Vadigepalli, Rajanikanth; Rafferty, Rachel; Gonye, Gregory E.; Weaver, David R.; Schwaber, James S.

    2012-01-01

    We use the multigenic pattern of gene expression across suprachiasmatic nuclei (SCN) regions and time to understand the dynamics within the SCN in response to a circadian phase-resetting light pulse. Global gene expression studies of the SCN indicate that circadian functions like phase resetting are complex multigenic processes. While the molecular dynamics of phase resetting are not well understood, it is clear they involve a “functional gene expression program”, e.g., the coordinated behavior of functionally related genes in space and time. In the present study we selected a set of 89 of these functionally related genes in order to further understand this multigenic program. By use of high-throughput qPCR we studied 52 small samples taken by anatomically precise laser capture from within the core and shell SCN regions, and taken at time points with and without phase resetting light exposure. The results show striking regional differences in light response to be present in the mouse SCN. By using network-based analyses, we are able to establish a highly specific multigenic correlation between genes expressed in response to light at night and genes normally activated during the day. The light pulse triggers a complex and highly coordinated network of gene regulation. The largest differences marking neuroanatomical location are in transmitter receptors, and the largest time-dependent differences occur in clock-related genes. Nighttime phase resetting appears to recruit transcriptional regulatory processes normally active in the day. This program, or mechanism, causes the pattern of core region gene expression to transiently shift to become more like that of the shell region. PMID:22662235

  7. Identification of a set of genes showing regionally enriched expression in the mouse brain

    PubMed Central

    D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa LC; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven JM

    2008-01-01

    Background The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression. PMID:18625066

  8. RGmatch: matching genomic regions to proximal genes in omics data integration.

    PubMed

    Furió-Tarí, Pedro; Conesa, Ana; Tarazona, Sonia

    2016-11-22

    The integrative analysis of multiple genomics data often requires that genome coordinates-based signals have to be associated with proximal genes. The relative location of a genomic region with respect to the gene (gene area) is important for functional data interpretation; hence algorithms that match regions to genes should be able to deliver insight into this information. In this work we review the tools that are publicly available for making region-to-gene associations. We also present a novel method, RGmatch, a flexible and easy-to-use Python tool that computes associations either at the gene, transcript, or exon level, applying a set of rules to annotate each region-gene association with the region location within the gene. RGmatch can be applied to any organism as long as genome annotation is available. Furthermore, we qualitatively and quantitatively compare RGmatch to other tools. RGmatch simplifies the association of a genomic region with its closest gene. At the same time, it is a powerful tool because the rules used to annotate these associations are very easy to modify according to the researcher's specific interests. Some important differences between RGmatch and other similar tools already in existence are RGmatch's flexibility, its wide range of user options, compatibility with any annotatable organism, and its comprehensive and user-friendly output.

  9. Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

    PubMed Central

    Bell, G I; Pictet, R; Rutter, W J

    1980-01-01

    The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes. Images PMID:6253909

  10. [Identification of 23 mycobacterial species by Invader assay with targeting 16S rRNA gene and ITS-1 region--comparison with DDH method in clinical isolates].

    PubMed

    Nagano, Makoto; Ichimura, Sadahiro; Ito, Nobuko; Tomii, Takayuki; Kazumi, Yuko; Takei, Katsuaki; Abe, Chiyoji; Sugawara, Isamu

    2008-07-01

    The Invader assay was developed to identify 23 mycobacterial species using probes derived from the species-specific region of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer 1 (ITS-1) region, with minor modifications of our previous study. In the present study, we compared the identification capability between the Invader assay and DNA-DNA hybridization (DDH) method. DDH is commonly used to identify non-tuberculosis mycobacterium in Japan and 636 clinical mycobacterial strains cultured on Ogawa slants were tested. The Invader assay could identify 615 (96.7%) of the 636 strains. The results contained 14 M.lentiflavum, 3 M. parascrofulaceum and 1 M. intermedium, which were undetectable with DDH method. On the other hand, DDH method could identify 580 (91.2%) strains with duplicate assay. Of 628 strains except 8 strains identified as a few species by Invader assay, 551 (87.7%) strains were identified as the same species by two methods. Discordant results were mainly recognized for the identification of M. gordonae, M. avium, M. lentiflavum and M. intracellurare. The results of other methods targeting 16S rRNA indicated correctness of the Invader assay. These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.

  11. The non-signaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity

    PubMed Central

    Hudecek, Michael; Sommermeyer, Daniel; Kosasih, Paula L.; Silva-Benedict, Anne; Liu, Lingfeng; Rader, Christoph; Jensen, Michael C.; Riddell, Stanley R.

    2015-01-01

    The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor-bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T-cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity. PMID:25212991

  12. The human VK locus. Characterization of a duplicated region encoding 28 different immunoglobulin genes.

    PubMed

    Straubinger, B; Huber, E; Lorenz, W; Osterholzer, E; Pargent, W; Pech, M; Pohlenz, H D; Zimmer, F J; Zachau, H G

    1988-01-05

    Two large regions of the human multigene family coding for the variable parts of the immunoglobulin light chains of the K type (VK) have been characterized on cosmid clones. The two germline regions, called Aa and Ab, span together 250,000 base-pairs and comprise 28 different VK gene segments, nine of which have been sequenced. There is a preponderance of VKII genes but genes belonging to subgroups I and III, and genes that cannot be easily assigned to one of the known subgroups, are interspersed within the VKII gene clusters. A number of pseudogenes have been identified. Within the Aa and Ab regions, all gene segments are organized in the same transcriptional orientation. The regions Aa and Ab, whose restriction maps are highly homologous, were shown not to be allelic structures; they must have arisen by a duplication event. Taken together with previous results, one can conclude that the major part of the VK locus exists in duplicated form. One individual has been found who has only one copy of some of the duplicated regions. By chromosomal walking, the A regions could be linked to the O regions, an analysis of which has been reported. The A regions contribute about one-third of the VK genes so far identified.

  13. Postfusion magnetic resonance imaging artifacts caused by a titanium, cobalt-chromium-molybdenum, and carbon intervertebral disc spacer.

    PubMed

    Ernstberger, Thorsten; Heidrich, Gabert

    2007-04-01

    Intervertebral spacers for anterior spine fusion are made of different materials, such as titanium and CoCrMo-alloys or carbon fiber reinforced polymers (CFRP). Implant-related susceptibility artifacts can decrease the quality of magnetic resonance imaging (MRI) scans. This cadaveric study aimed to demonstrate the extent that implant-related MRI artifacting affects the postfusion differentiation of the spinal canal (SC) and intervertebral disc space (IDS). In 6 cadaveric porcine spines, we evaluated the postimplantation MRI scans of a titanium, CoCrMo-spacer and CFRP-spacer that differed in shape and surface qualities. A spacer made of human cortical bone was used as a control. A defined evaluation unit was divided into regions of interest (ROI) to characterize the SC and IDS. Considering 15 different MRI sequences read independently by an interobserver-validated team of specialists artifact-affected image quality of the median MRI slice was rated on a score of 0-1-2-3. A maximum score of 15 points for the SC and 9 points for the IDS (100%) was possible. Turbo spin echo sequences produced the best scores for both spacers and the control. Only the control achieved a score of 100%. For the IDS the CoCrMo-spacer, titanium and CFRP-spacer maximally scored 0%, 0% and 74%, for the SC 60%, 80% and 99%, respectively. By using favored T1 TSE sequences the CFRP-spacer represented clear advantages in postfusion spinal imaging. Independent of artifact dimensions the used scoring system allowed us to create an implant-related ranking of MRI scan quality in reference to the bone control.

  14. Sequence analysis of the ERCC2 gene regions in human, mouse, and hamster reveals three linked genes.

    PubMed

    Lamerdin, J E; Stilwagen, S A; Ramirez, M H; Stubbs, L; Carrano, A V

    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' 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 cycle 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.

  15. 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.

  16. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using internal transcribed spacer and cytochrome oxidase subunit l DNA sequences

    USDA-ARS?s Scientific Manuscript database

    The genetic diversity of Tamarixia radiata laboratory colonies derived from collections in China, northern Vietnam, Pakistan, and a mixed colony from Taiwan and southern Vietnam was evaluated using the internal transcribed spacer region 1 (ITS-1), internal transcribed spacer region 2 (ITS-2) and the...

  17. Genetic diversity and phylogenetic analysis of Citrus (L) from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA

    PubMed Central

    Hynniewta, Marlykynti; Malik, Surendra Kumar; Rao, Satyawada Rama

    2014-01-01

    The north-eastern region of India is reported to be the center of origin and rich in diversity of Citrus (L.) species, where some wild and endangered species namely Citrus indica, Citrus macroptera, Citrus latipes, Citrus ichagensis and Citrus assamensis exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various Citrus species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs) analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of Citrus species confirming our cytogenetical interpretations. The three true basic species i.e. Citrus medica, Citrus maxima and Citrus reticulata with their unique status have been resolved into distinct clades with molecular approaches as well. C. indica which occupies a unique position in the phylogenetic ladder of the genus Citrus has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in C. indica also echo and support its unique position. From our study it is amply clear that C

  18. Genetic diversity and phylogenetic analysis of Citrus (L) from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA.

    PubMed

    Hynniewta, Marlykynti; Malik, Surendra Kumar; Rao, Satyawada Rama

    2014-12-01

    The north-eastern region of India is reported to be the center of origin and rich in diversity of Citrus (L.) species, where some wild and endangered species namely Citrus indica, Citrus macroptera, Citrus latipes, Citrus ichagensis and Citrus assamensis exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various Citrus species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs) analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of Citrus species confirming our cytogenetical interpretations. The three true basic species i.e. Citrus medica, Citrus maxima and Citrus reticulata with their unique status have been resolved into distinct clades with molecular approaches as well. C. indica which occupies a unique position in the phylogenetic ladder of the genus Citrus has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in C. indica also echo and support its unique position. From our study it is amply clear that C

  19. Development of an Intelligent Spacer Data Logger System.

    PubMed

    O'Callaghan, Chris; Smith, Nicholas J; Barry, Peter W; Denyer, John

    2017-08-28

    Although delivery of drugs from pressurized metered dose inhalers (pMDIs) via spacer devices is widespread it cannot be assumed that patients take their medication as prescribed or use their spacer appropriately. We developed a Spacer Data Logger device to record patient adherence and whether patients had shaken the pMDI, actuated it soon after shaking, and inhaled a sufficient volume from it. We report an assessment of the Spacer Data Logger to measure and record that the pMDI was adequately shaken, the time to actuation, and the volume "inhaled" from the spacer up to 26 seconds after actuation. The effect of a delay in actuation following shaking on the dose available for inhalation from the spacer and the effect of a delay in extraction of aerosol from the spacer were assessed using different strengths of beclomethasone dipropionate (50 and 100 μg) and fluticasone propionate (50, 125 and 250 μg). The volumes measured by the Spacer Data Logger were in close agreement with the reference volumes of four simulated breathing patterns. A delay between shaking and actuating the pMDI resulted in a significant increase in the dose available for inhalation after only 4 seconds for the 50 and 250 μg strengths of fluticasone propionate pMDIs (p = 0.004 and p < 0.001, respectively). A delay between actuation of the drug into the spacer and "inhalation" of aerosol from the spacer also resulted in a steady decline in the dose available from the spacer (p < 0.0001). These results confirmed the importance of using the pMDI spacer correctly by actuating directly after shaking and inhaling the aerosol from the spacer as soon after actuation as possible to optimize the dose available for inhalation. The Spacer Data Logger should be a useful tool to determine adherence to and "optimum" use of pMDI spacers in patients with asthma and chronic obstructive pulmonary disease (COPD).

  20. Alternating tandem array of histone and ribosomal RNA gene blocks in the boll weevil

    USDA-ARS?s Scientific Manuscript database

    Histones are the major protein component of the ncleosome. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and the clust...

  1. The involucrin genes of the white-fronted capuchin and cottontop tamarin: the platyrrhine middle region.

    PubMed

    Phillips, M; Rice, R H; Djian, P; Green, H

    1991-09-01

    In all anthropoid species, the coding region of the involucrin gene contains a segment of short tandem repeats that were added sequentially, beginning in a common anthropoid ancestor. The involucrin coding region of each of two platyrrhine species, the white-fronted capuchin (Cebus albifrons) and the cottontop tamarin (Saguinus oedipus), has now been cloned and sequenced. These genes share with the genes of the catarrhines the repeats added in the common anthropoid lineage (the early region). After their divergence, the platyrrhines, like the catarrhines, continued to add repeats vectorially 5' of the early region, to form a middle region. The mechanism that was established in the common anthropoid lineage for the addition of repeats at a definite site in the coding region was transmitted to both platyrrhines and catarrhines, enabling each to generate its middle region independently. The process of vectorial repeat addition continued in two platyrrhine sublineages after their divergence from each other.

  2. Low abundant spacer 5S rRNA transcripts are frequently polyadenylated in Nicotiana.

    PubMed

    Fulnecek, Jaroslav; Kovarik, Ales

    2007-11-01

    In plants, 5S rRNA genes (5S rDNA) encoding 120-nt structural RNA molecules of ribosomes are organized in tandem arrays comprising thousands of units. Failure to correctly terminate transcription would generate longer inaccurately processed transcripts interfering with ribosome biogenesis. Hence multiple termination signals occur immediately after the 5S rRNA coding sequence. To obtain information about the efficiency of termination of 5S rDNA transcription in plants we analyzed 5S rRNA pools in three Nicotiana species, N. sylvestris, N. tomentosiformis and N. tabacum. In addition to highly abundant 120-nt 5S rRNA transcripts, we also detected RNA species composed of a genic region and variable lengths of intergenic sequences. These genic-intergenic RNA molecules occur at a frequency severalfold lower than the mature 120-nt transcripts, and are posttranscriptionally modified by polyadenylation at their 3' end in contrast to 120-nt transcripts. An absence of 5S small RNAs (smRNA) argue against a dominant role for the smRNA biosynthesis pathway in the degradation of aberrant 5S rRNA in Nicotiana. This work is the first description of polyadenylated 5S rRNA species in higher eukaryotes originating from a read-through transcription into the intergenic spacer. We propose that polyadenylation may function in a "quality control" pathway ensuring that only correctly processed molecules enter the ribosome biogenesis.

  3. DNA regions that regulate the ovarian transcriptional specificity of Drosophila yolk protein genes.

    PubMed

    Logan, S K; Garabedian, M J; Wensink, P C

    1989-09-01

    Yolk protein genes 1 and 2 (yp1 and yp2) of Drosophila melanogaster are divergently transcribed neighboring genes. Both are transcribed in only two tissues, the ovarian follicle cells and the fat bodies of adult females. Previous work has identified a yolk protein enhancer between the genes that is sufficient to direct transcription in one of the tissues, female fat bodies. Using germ-line transformation methods, we identify two cis-acting regions with positive effects on transcription in ovaries. One, a 301-bp region located between the genes, influences both genes and is an enhancer determining the stage and cell type specificity of ovarian transcription. The other, a 105-bp region located in the first exon of yp2, acts across the yp2 promoter region to stimulate yp1 transcription in ovaries. Additional observations suggest how a single enhancer influences both promoters.

  4. Regional and strain-specific gene expression mapping in the adult mouse brain

    PubMed Central

    Sandberg, Rickard; Yasuda, Rie; Pankratz, Daniel G.; Carter, Todd A.; Del Rio, Jo A.; Wodicka, Lisa; Mayford, Mark; Lockhart, David J.; Barlow, Carrolee

    2000-01-01

    To determine the genetic causes and molecular mechanisms responsible for neurobehavioral differences in mice, we used highly parallel gene expression profiling to detect genes that are differentially expressed between the 129SvEv and C57BL/6 mouse strains at baseline and in response to seizure. In addition, we identified genes that are differentially expressed in specific brain regions. We found that approximately 1% of expressed genes are differentially expressed between strains in at least one region of the brain and that the gene expression response to seizure is significantly different between the two inbred strains. The results lead to the identification of differences in gene expression that may account for distinct phenotypes in inbred strains and the unique functions of specific brain regions. PMID:11005875

  5. Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes.

    PubMed Central

    Barlow, J W; Voz, M L; Eliard, P H; Mathy-Harter, M; De Nayer, P; Economidis, I V; Belayew, A; Martial, J A; Rousseau, G G

    1986-01-01

    The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions. PMID:3466175

  6. Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes.

    PubMed

    Barlow, J W; Voz, M L; Eliard, P H; Mathy-Harter, M; De Nayer, P; Economidis, I V; Belayew, A; Martial, J A; Rousseau, G G

    1986-12-01

    The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions.

  7. Chronic ethanol exposure produces time- and brain region-dependent changes in gene coexpression networks.

    PubMed

    Osterndorff-Kahanek, Elizabeth A; Becker, Howard C; Lopez, Marcelo F; Farris, Sean P; Tiwari, Gayatri R; Nunez, Yury O; Harris, R Adron; Mayfield, R Dayne

    2015-01-01

    Repeated ethanol exposure and withdrawal in mice increases voluntary drinking and represents an animal model of physical dependence. We examined time- and brain region-dependent changes in gene coexpression networks in amygdala (AMY), nucleus accumbens (NAC), prefrontal cortex (PFC), and liver after four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure in C57BL/6J mice. Microarrays were used to compare gene expression profiles at 0-, 8-, and 120-hours following the last ethanol exposure. Each brain region exhibited a large number of differentially expressed genes (2,000-3,000) at the 0- and 8-hour time points, but fewer changes were detected at the 120-hour time point (400-600). Within each region, there was little gene overlap across time (~20%). All brain regions were significantly enriched with differentially expressed immune-related genes at the 8-hour time point. Weighted gene correlation network analysis identified modules that were highly enriched with differentially expressed genes at the 0- and 8-hour time points with virtually no enrichment at 120 hours. Modules enriched for both ethanol-responsive and cell-specific genes were identified in each brain region. These results indicate that chronic alcohol exposure causes global 'rewiring' of coexpression systems involving glial and immune signaling as well as neuronal genes.

  8. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer

    PubMed Central

    Richter, Corinna; Dy, Ron L.; McKenzie, Rebecca E.; Watson, Bridget N.J.; Taylor, Corinda; Chang, James T.; McNeil, Matthew B.; Staals, Raymond H.J.; Fineran, Peter C.

    2014-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary. PMID:24990370

  9. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

    PubMed

    Richter, Corinna; Dy, Ron L; McKenzie, Rebecca E; Watson, Bridget N J; Taylor, Corinda; Chang, James T; McNeil, Matthew B; Staals, Raymond H J; Fineran, Peter C

    2014-07-01

    Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

  10. Regional Expression of MTG Genes in the Developing Mouse Central Nervous System

    PubMed Central

    Alishahi, Amin; Koyano-Nakagawa, Naoko; Nakagawa, Yasushi

    2009-01-01

    Myeloid translocation gene (MTG) proteins are transcriptional repressors that are highly conserved across species. We studied the expression of three members of this gene family, MTGR1, MTG8, and MTG16 in developing mouse central nervous system by in situ hybridization. All of these genes are detected as early as embryonic day 11.5. Because these genes are known to be induced by proneural genes during neurogenesis, we analyzed the expression of MTG genes in relation to two proneural genes, Neurog2 (also known as Ngn2 or Neurogenin 2) and Ascl1 (also known as Mash1). While MTGR1 are generally expressed in regions that also express Neurog2, MTG8 and MTG16 expression is associated more tightly with that of Ascl1-expressing neural progenitor cells. These results suggest the possibility that expression of MTG genes is differentially controlled by specific proneural genes during neurogenesis. PMID:19618476

  11. Strong early seed-specific gene regulatory region

    DOEpatents

    Broun, Pierre; Somerville, Chris

    2002-01-01

    Nucleic acid sequences and methods for their use are described which provide for early seed-specific transcription, in order to modulate or modify expression of foreign or endogenous genes in seeds, particularly embryo cells. The method finds particular use in conjunction with modifying fatty acid production in seed tissue.

  12. Strong early seed-specific gene regulatory region

    DOEpatents

    Broun, Pierre; Somerville, Chris

    1999-01-01

    Nucleic acid sequences and methods for their use are described which provide for early seed-specific transcription, in order to modulate or modify expression of foreign or endogenous genes in seeds, particularly embryo cells. The method finds particular use in conjunction with modifying fatty acid production in seed tissue.

  13. Spatially differentiated expression of quadruplicated green-sensitive RH2 opsin genes in zebrafish is determined by proximal regulatory regions and gene order to the locus control region.

    PubMed

    Tsujimura, Taro; Masuda, Ryoko; Ashino, Ryuichi; Kawamura, Shoji

    2015-11-04

    Fish are remarkably diverse in repertoires of visual opsins by gene duplications. Differentiation of their spatiotemporal expression patterns and absorption spectra enables fine-tuning of feature detection in spectrally distinct regions of the visual field during ontogeny. Zebrafish have quadruplicated green-sensitive (RH2) opsin genes in tandem (RH2-1, -2, -3, -4), which are expressed in the short member of the double cones (SDC). The shortest wavelength RH2 subtype (RH2-1) is expressed in the central to dorsal area of the adult retina. The second shortest wave subtype (RH2-2) is expressed overlapping with RH2-1 but extending outside of it. The second longest wave subtype (RH2-3) is expressed surrounding the RH2-2 area, and the longest wave subtype (RH2-4) is expressed outside of the RH2-3 area broadly occupying the ventral area. Expression of the four RH2 genes in SDC requires a single enhancer (RH2-LCR), but the mechanism of their spatial differentiation remains elusive. Functional comparison of the RH2-LCR with its counterpart in medaka revealed that the regulatory role of the RH2-LCR in SDC-specific expression is evolutionarily conserved. By combining the RH2-LCR and the proximal upstream region of each RH2 gene with fluorescent protein reporters, we show that the RH2-LCR and the RH2-3 proximal regulatory region confer no spatial selectivity of expression in the retina. But those of RH2-1, -2 and -4 are capable of inducing spatial differentiation of expression. Furthermore, by analyzing transgenic fish with a series of arrays consisting of the RH2-LCR and multiple upstream regions of the RH2 genes in different orders, we show that a gene expression pattern related to an upstream region is greatly influenced by another flanking upstream region in a relative position-dependent manner. The zebrafish RH2 genes except RH2-3 acquired differential cis-elements in the proximal upstream regions to specify the differential expression patterns. The input from these

  14. Synonymous Substitutions in the Xdh Gene of Drosophila: Heterogeneous Distribution along the Coding Region

    PubMed Central

    Comeron, J. M.; Aguade, M.

    1996-01-01

    The Xdh (rosy) region of Drosophila subobscura has been sequenced and compared to the homologous region of D. pseudoobscura and D. melanogaster. Estimates of the numbers of synonymous substitutions per site (Ks) confirm that Xdh has a high synonymous substitution rate. The distributions of both nonsynonymous and synonymous substitutions along the coding region were found to be heterogeneous. Also, no relationship has been detected between Ks estimates and codon usage bias along the gene, in contrast with the generally observed relationship among genes. This heterogeneous distribution of synonymous substitutions along the Xdh gene, which is expression-level independent, could be explained by a differential selection pressure on synonymous sites along the coding region acting on mRNA secondary structure. The synonymous rate in the Xdh coding region is lower in the D. subobscura than in the D. pseudoobscura lineage, whereas the reverse is true for the Adh gene. PMID:8913749

  15. Molecular recordings by directed CRISPR spacer acquisition.

    PubMed

    Shipman, Seth L; Nivala, Jeff; Macklis, Jeffrey D; Church, George M

    2016-07-29

    The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution so as to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.

  16. Mask specification guidelines in spacer patterning technology

    NASA Astrophysics Data System (ADS)

    Hashimoto, Kohji; Mukai, Hidefumi; Miyoshi, Seiro; Yamaguchi, Shinji; Mashita, Hiromitsu; Kobayashi, Yuuji; Kawano, Kenji; Hirano, Takashi

    2008-11-01

    We have studied both the mask CD specification and the mask defect specification for spacer patterning technology (SPT). SPT has the possibility of extending optical lithography to below 40nm half-pitch devices. Since SPT necessitates somewhat more complicated wafer process flow, the CD error and mask defect printability on wafers involve more process factors compared with conventional single-exposure process (SEP). This feature of SPT implies that it is very important to determine mask-related specifications for SPT in order to select high-end mask fabrication strategies; those are for mask writing tools, mask process development, materials, inspection tools, and so on. Our experimental studies reveal that both mask CD specification and mask defect specification are somehow relaxed from those in ITRS2007. This is most likely because SPT reduces mask CD error enhanced factor (MEF) and the reduction of line-width roughness (LWR).

  17. Molecular recordings by directed CRISPR spacer acquisition

    PubMed Central

    Shipman, Seth L; Nivala, Jeff; Macklis, Jeffrey D; Church, George M

    2016-01-01

    The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR-Cas system of E. coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device. PMID:27284167

  18. Medpor lower eyelid spacer: does it biointegrate?

    PubMed

    Mavrikakis, Ioannis; Francis, Nick; Poitelea, Cornelia; Parkin, Ben; Brittain, Paul; Olver, Jane

    2009-01-01

    To report the histopathologic findings of explanted Medpor lower eyelid spacers (LES) in complicated cases. Four cases of lower eyelid retraction due to thyroid orbitopathy (n = 2), facial nerve palsy (n = 1), and post-enucleation socket syndrome (n = 1) were treated with Medpor LES. All implants were removed between 6 months to 2 years following their original insertion due to exposure, poor stability, or contour. Histopathology of the implants showed fibrosis and vascularization although clinically, at the time of removal, did not appear vascularized. In addition, immunohistochemistry was positive for Factor VIII related antigen and CD34, thus highlighting the presence of vessels in the pores and around the implant. To our knowledge, we are the first to report histopathologic findings of explanted high-density porous polyethylene implants from the lower eyelid in humans. Although this study shows that Medpor LES does biointegrate, we advocate using it sparingly due to associated complications such as exposure, poor stability, and contour.

  19. A 5′- Regulatory Region and Two Coding Region Polymorphisms Modulate Promoter Activity and Gene Expression of the Growth Suppressor Gene ZBED6 in Cattle

    PubMed Central

    Huang, Yong-Zhen; Li, Ming-Xun; Wang, Jing; Zhan, Zhao-Yang; Sun, Yu-Jia; Sun, Jia-Jie; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Chen, Hong

    2013-01-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. Polymorphisms in its promoter and coding regions are likely to impact ZBED6 transcription and growth traits. In this study, rapid amplification of 5’ cDNA ends (5'-RACE) analysis revealed two transcription start sites (TSS) for the bovine ZBED6 starting within exon 1 of the ZC3H11A gene (TSS-1) and upstream of the translation start codon of the ZBED6 gene (TSS-2). There was one SNP in the promoter and two missense mutations in the coding region of the bovine ZBED6 by sequencing of the pooled DNA samples (Pool-Seq, n = 100). The promoter and coding region are the key regions for gene function; polymorphisms in these regions can alter gene expression. Quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution in cattle and is highly expressed in skeletal muscle. Eleven promoter-detection vectors were constructed, which enabled the cloning of putative promoter sequences and analysis of ZBED6 transcriptional activity by luciferase reporter gene assays. The core region of the basal promoter of bovine ZBED6 is located within region -866 to -556. The activity of WT-826G-pGL3 in driving reporter gene transcription is significantly higher than that of the M-826A-pGL3 construct (P < 0.01). Analysis of gene expression patterns in homozygous full-sibling Chinese Qinchuan cattle showed that the mutant-type Hap-AGG exhibited a lower mRNA level than the wild-type Hap-GCA (P < 0.05) in longissimus dorsi muscle (LDM). Moreover, ZBED6 mRNA expression was low in C2C12 cells overexpressing the mutant-type ZBED6 (pcDNA3.1+-Hap-GG) (P < 0.01). Our results suggest that the polymorphisms in the promoter and coding regions may modulate the promoter activity and gene expression of bovine ZBED6 in the skeletal muscles of these cattle breeds. PMID:24223190

  20. Targeting drugs to the airways: The role of spacer devices.

    PubMed

    Lavorini, Federico; Fontana, Giovanni A

    2009-01-01

    Spacer devices are inhalation aids of varying dimension and complexity, specifically designed to overcome problems with the use of pressurised metered dose inhalers (pMDIs). The aim of this review is to examine the current understanding about these inhalation devices and discuss their advantages and disadvantages. The pertinent literature concerning the characteristics and effects of spacers on delivery and lung deposition of inhaled medications, as well as their clinical efficacy in patients with reversible airway obstruction, is examined. Spacers minimise problems of poor inhalation technique with pMDI, reduce oropharyngeal deposition and increase lung deposition. Spacers improve the clinical effect of inhaled medications, especially in patients unable to use a pMDI properly. Compared to both pMDIs and dry-powder inhalers, spacers may increase the response to beta-adrenergic bronchodilators, even in patients with correct inhalation technique. A pMDI plus spacer has proven to be viable lower cost alternative to the use of a nebuliser for delivering large bronchodilator doses in patients with severe acute asthma or chronic obstructive pulmonary disease. The use of large-volume spacers is recommended for delivering high doses of inhaled corticosteroids, and may permit a lower maintenance dose to be used. pMDIs may be routinely fitted with a spacer, especially in situations where correct pMDI use is unlikely.

  1. Chloroplast Genome Differences between Asian and American Equisetum arvense (Equisetaceae) and the Origin of the Hypervariable trnY-trnE Intergenic Spacer

    PubMed Central

    Kim, Hyoung Tae; Kim, Ki-Joong

    2014-01-01

    Comparative analyses of complete chloroplast (cp) DNA sequences within a species may provide clues to understand the population dynamics and colonization histories of plant species. Equisetum arvense (Equisetaceae) is a widely distributed fern species in northeastern Asia, Europe, and North America. The complete cp DNA sequences from Asian and American E. arvense individuals were compared in this study. The Asian E. arvense cp genome was 583 bp shorter than that of the American E. arvense. In total, 159 indels were observed between two individuals, most of which were concentrated on the hypervariable trnY-trnE intergenic spacer (IGS) in the large single-copy (LSC) region of the cp genome. This IGS region held a series of 19 bp repeating units. The numbers of the 19 bp repeat unit were responsible for 78% of the total length difference between the two cp genomes. Furthermore, only other closely related species of Equisetum also show the hypervariable nature of the trnY-trnE IGS. By contrast, only a single indel was observed in the gene coding regions: the ycf1 gene showed 24 bp differences between the two continental individuals due to a single tandem-repeat indel. A total of 165 single-nucleotide polymorphisms (SNPs) were recorded between the two cp genomes. Of these, 52 SNPs (31.5%) were distributed in coding regions, 13 SNPs (7.9%) were in introns, and 100 SNPs (60.6%) were in intergenic spacers (IGS). The overall difference between the Asian and American E. arvense cp genomes was 0.12%. Despite the relatively high genetic diversity between Asian and American E. arvense, the two populations are recognized as a single species based on their high morphological similarity. This indicated that the two regional populations have been in morphological stasis. PMID:25157804

  2. Short 5'-flanking regions of the Amy gene of Drosophila kikkawai affect amylase gene expression and respond to food environments.

    PubMed

    Inomata, Nobuyuki; Nakashima, Shuichi

    2008-04-15

    Evolution of the duplicated genes and regulation in gene expression is of great interest, especially in terms of adaptation. Molecular population genetic and evolutionary studies on the duplicated amylase genes of Drosophila species have suggested that their 5'-flanking (cis-regulatory) regions play an important role in evolution of these genes. For better understanding of evolution of the duplicated amylase genes and gene expression, we studied functional significance of the Amy1 gene of Drosophila kikkawai using in vitro deletion mutagenesis followed by P-element-mediated germline transformation. We found that a 1.6-kb of the 5'-flanking region can produce strikingly higher level of larval amylase activity on starch food compared with that on glucose food. We found two cis-regulatory elements, which increase larval amylase activity on starch food. We also found a larval cis-regulatory element, which responds to the food difference. This food-response element is necessary for the function of the element increasing larval activity on starch food. A 5-bp deletion in a putative GRE caused high amylase activity, indicating a cis-regulatory element decreasing amylase activity. These cis-regulatory elements identified in the 5'-flanking region could be the targets of natural selection.

  3. PCR primers for 30 novel gene regions in the nuclear genomes of Lepidoptera.

    PubMed

    Wahlberg, Niklas; Peña, Carlos; Ahola, Milla; Wheat, Christopher W; Rota, Jadranka

    2016-01-01

    We report primer pairs for 30 new gene regions in the nuclear genomes of Lepidoptera that can be amplified using a standard PCR protocol. The new primers were tested across diverse Lepidoptera, including nonditrysians and a wide selection of ditrysians. These new gene regions give a total of 11,043 bp of DNA sequence data and they show similar variability to traditionally used nuclear gene regions in studies of Lepidoptera. We feel that a PCR-based approach still has its place in molecular systematic studies of Lepidoptera, particularly at the intrafamilial level, and our new set of primers now provides a route to generating phylogenomic datasets using traditional methods.

  4. The Characterisation of Three Types of Genes that Overlie Copy Number Variable Regions

    PubMed Central

    Woodwark, Cara; Bateman, Alex

    2011-01-01

    Background Due to the increased accuracy of Copy Number Variable region (CNV) break point mapping, it is now possible to say with a reasonable degree of confidence whether a gene (i) falls entirely within a CNV; (ii) overlaps the CNV or (iii) actually contains the CNV. We classify these as type I, II and III CNV genes respectively. Principal Findings Here we show that although type I genes vary in copy number along with the CNV, most of these type I genes have the same expression levels as wild type copy numbers of the gene. These genes must, therefore, be under homeostatic dosage compensation control. Looking into possible mechanisms for the regulation of gene expression we found that type I genes have a significant paucity of genes regulated by miRNAs and are not significantly enriched for monoallelically expressed genes. Type III genes, on the other hand, have a significant excess of genes regulated by miRNAs and are enriched for genes that are monoallelically expressed. Significance Many diseases and genomic disorders are associated with CNVs so a better understanding of the different ways genes are associated with normal CNVs will help focus on candidate genes in genome wide association studies. PMID:21637334

  5. Identification of Disease-Transmitting Mosquitoes: Development of Species-Specific Probes for DNA Chip Assay Using Mitochondrial COI and ND2 Genes and Ribosomal Internal Transcribed Spacer 2.

    PubMed

    Wang, Xi; Tu, Wu-Chun; Huang, En-Jiong; Chen, Yen-Hou; Chen, Jia-Hua; Yeh, Wen-Bin

    2017-03-01

    Mosquitoes, which transmit infectious diseases, such as malaria and dengue fever, are harmful to human health. Thus, accurate and rapid identification of vectors is a critical step for the control of mosquito-borne diseases. However, phenotypic variations in adults, lack of recognizable features of the immature, and fragility of mosquitoes make identification difficult. Molecular approaches have been widely applied to identify mosquitoes, yet these methods have been focused only on the identification of a few species. This study used sequences of two mitochondrial genes, COI and ND2, and a ribosomal gene, ITS2, to design species-specific probes. Biochips thus developed were able to provide simultaneous identification of nine important medical and veterinary species, including the immature, from genera of Aedes, Anopheles, Armigeres, and Culex. This chip was also applied to samples collected from the field. Despite its inability to resolve the close affinity species of Culex quinquefasciatus and Culex pipiens molestus, pertinent biochips are expected to be applied to a mass screening method. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Analysis of the sexual development-promoting region of Schizophyllum commune TRP1 gene.

    PubMed

    Sen, Kikuo; Kinoshita, Hideki; Tazuke, Kazuyuki; Maki, Yoshinori; Yoshiura, Yumi; Yakushi, Toshiharu; Shibai, Hiroshiro; Kurosawa, Shin-Ichi

    2016-10-01

    This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp(-)) had a missense mutation (L→F) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.

  7. Isolation and characterization of candidate genes of the 5q13 region in spinal muscular atrophy

    SciTech Connect

    Lefebvre, S.; Reboullet, S.; Benichou, B.

    1994-09-01

    Based on a fine genetic and physical map of the region deleted in spinal muscular atrophy, we defined the smallest rearrangements encompassing the SMA gene. This interval is entirely contained in the 903D1 YAC clone. Several approaches to identify candidate genes were applied, including the search for interspecies conservation, exon trapping amplification and direct cDNA selection. Combining these strategies, six different cDNA molecules mapping to the YAC contig were isolated. Four cDNA molecules were isolated using the exon trapping system. They map to chromosome 5p and to more than one locus within the 5q13 region. They are homologous to each other and share sequence homology with the {beta}-glucuronidase gene. Based on interspecies conservation, a fifth candidate gene was identified. Sequence analyses of the cDNAs revealed no homologies with any other described genes. This gene mapped to two loci within the 5q13 region. Two other cDNA molecules isolated by direct cDNA selection are also under investigation. Complete characterization and fine physical mapping of those genes with respect to the physical interval defined by the deletions of the SMA region will allow the identification of the disease gene (or genes).

  8. A non-electrostatic spacer for aerosol delivery.

    PubMed Central

    Bisgaard, H; Anhøj, J; Klug, B; Berg, E

    1995-01-01

    A pear shaped non-electrostatic spacer, composed of steel with a volume of 250 ml and equipped with a facemask containing integrated inlet and outlet valves for inspiration and expiration, was compared with three plastic spacers. The plastic spacers were primed with repeated puffs from a budesonide pressurised metered dose inhaler (p-MDI) to minimise the electrostatic charge on the plastic. The procedure prolonged the half life (t1/2) of the aerosol in the Nebuhaler from nine to 32 seconds. A normal cleaning procedure reduced the aerosol t1/2 back to baseline. The t1/2 of the aerosol in the metal spacer was 27 seconds and independent of the use of p-MDI. In vitro the maximum dose of budesonide from a p-MDI, expressed as a percentage of the nominal dose, was 56% from the non-electrostatic spacer, 61% from the Nebuhaler, 45% from the Babyhaler, and 30% from the AeroChamber. In 124 children, age 6 months to 6 years, suspected to have asthma the non-electrostatic spacer delivered a mean total dose of budesonide aerosol of 39% of the nominal dose, which was significantly higher than the Babyhaler (28%), the Nebuhaler (21%), and the AeroChamber (19%). These differences were most pronounced in children younger than 4 years. The improved dose delivery from the small volume non-electrostatic spacer is probably related to the non-electrostatic spacer material and the valves which assured unidirectional airflow from the spacer without adding any dead space in the inspiratory channel. The non-electro-static spacer should improve the cost effectiveness of aerosol treatment and, as the counteracting effects of proming and recharging of the plastic from cleaning are avoided, should deliver a more reliable dose. PMID:7492160

  9. Complex repetitive arrangements of gene sequence in the candidate region of the spinal muscular atrophy gene in 5q13

    SciTech Connect

    Theodosiou, A.M.; Nesbit, A.M.; Daniels, R.J.; Campbell, L.; Francis, M.J.; Christodoulou, Z.; Morrison, K.E.; Davies, K.E. |

    1994-12-01

    Childhood-onset proximal spinal muscular atrophy (SMA) is a heritable neurological disorder, which has been mapped by genetic linkage analysis to chromosome 5q13, in the interval between markers D5S435 and D5S557. Here, we present gene sequences that have been isolated from this interval, several of which show sequence homologies to exons of {beta}-glucuronidase. These gene sequences are repeated several times across the candidate region and are also present on chromosome 5p. The arrangement of these repetitive gene motifs is polymorphic between individuals. The high degree of variability observed may have some influence on the expression of the genes in the region. Since SMA is not inherited as a classical autosomal recessive disease, novel genomic rearrangements arising from aberrant recombination events between the complex repeats may be associated with the phenotype observed.

  10. Single nucleotide polymorphism in transcriptional regulatory regions and expression of environmentally responsive genes

    SciTech Connect

    Wang, Xuting; Tomso, Daniel J.; Liu Xuemei; Bell, Douglas A. . E-mail: BELL1@niehs.nih.gov

    2005-09-01

    Single nucleotide polymorphisms (SNPs) in the human genome are DNA sequence variations that can alter an individual's response to environmental exposure. SNPs in gene coding regions can lead to changes in the biological properties of the encoded protein. In contrast, SNPs in non-coding gene regulatory regions may affect gene expression levels in an allele-specific manner, and these functional polymorphisms represent an important but relatively unexplored class of genetic variation. The main challenge in analyzing these SNPs is a lack of robust computational and experimental methods. Here, we first outline mechanisms by which genetic variation can impact gene regulation, and review recent findings in this area; then, we describe a methodology for bioinformatic discovery and functional analysis of regulatory SNPs in cis-regulatory regions using the assembled human genome sequence and databases on sequence polymorphism and gene expression. Our method integrates SNP and gene databases and uses a set of computer programs that allow us to: (1) select SNPs, from among the >9 million human SNPs in the NCBI dbSNP database, that are similar to cis-regulatory element (RE) consensus sequences; (2) map the selected dbSNP entries to the human genome assembly in order to identify polymorphic REs near gene start sites; (3) prioritize the candidate polymorphic RE containing genes by searching the existing genotype and gene expression data sets. The applicability of this system has been demonstrated through studies on p53 responsive elements and is being extended to additional pathways and environmentally responsive genes.

  11. Intercalation of XR5944 with the estrogen response element is modulated by the tri-nucleotide spacer sequence between half-sites.

    PubMed

    Sidell, Neil; Mathad, Raveendra I; Shu, Feng-jue; Zhang, Zhenjiang; Kallen, Caleb B; Yang, Danzhou

    2011-04-01

    DNA-intercalating molecules can impair DNA replication, DNA repair, and gene transcription. We previously demonstrated that XR5944, a DNA bis-intercalator, specifically blocks binding of estrogen receptor-α (ERα) to the consensus estrogen response element (ERE). The consensus ERE sequence is AGGTCAnnnTGACCT, where nnn is known as the tri-nucleotide spacer. Recent work has shown that the tri-nucleotide spacer can modulate ERα-ERE binding affinity and ligand-mediated transcriptional responses. To further understand the mechanism by which XR5944 inhibits ERα-ERE binding, we tested its ability to interact with consensus EREs with variable tri-nucleotide spacer sequences and with natural but non-consensus ERE sequences using one dimensional nuclear magnetic resonance (1D (1)H NMR) titration studies. We found that the tri-nucleotide spacer sequence significantly modulates the binding of XR5944 to EREs. Of the sequences that were tested, EREs with CGG and AGG spacers showed the best binding specificity with XR5944, while those spaced with TTT demonstrated the least specific binding. The binding stoichiometry of XR5944 with EREs was 2:1, which can explain why the spacer influences the drug-DNA interaction; each XR5944 spans four nucleotides (including portions of the spacer) when intercalating with DNA. To validate our NMR results, we conducted functional studies using reporter constructs containing consensus EREs with tri-nucleotide spacers CGG, CTG, and TTT. Results of reporter assays in MCF-7 cells indicated that XR5944 was significantly more potent in inhibiting the activity of CGG- than TTT-spaced EREs, consistent with our NMR results. Taken together, these findings predict that the anti-estrogenic effects of XR5944 will depend not only on ERE half-site composition but also on the tri-nucleotide spacer sequence of EREs located in the promoters of estrogen-responsive genes.

  12. A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes

    PubMed Central

    Sahoo, Dipak K.; Abeysekara, Nilwala S.; Cianzio, Silvia R.; Robertson, Alison E.

    2017-01-01

    Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance. PMID:28081566

  13. A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes.

    PubMed

    Sahoo, Dipak K; Abeysekara, Nilwala S; Cianzio, Silvia R; Robertson, Alison E; Bhattacharyya, Madan K

    2017-01-01

    Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

  14. Spacer effect on nanostructures and self-assembly in organogels via some bolaform cholesteryl imide derivatives with different spacers

    NASA Astrophysics Data System (ADS)

    Jiao, Tifeng; Gao, Fengqing; Zhang, Qingrui; Zhou, Jingxin; Gao, Faming

    2013-10-01

    In this paper, new bolaform cholesteryl imide derivatives with different spacers were designed and synthesized. Their gelation behaviors in 23 solvents were investigated, and some of them were found to be low molecular mass organic gelators. The experimental results indicated that these as-formed organogels can be regulated by changing the flexible/rigid segments in spacers and organic solvents. Suitable combination of flexible/rigid segments in molecular spacers in the present cholesteryl gelators is favorable for the gelation of organic solvents. Scanning electron microscopy and atomic force microscopy observations revealed that the gelator molecules self-assemble into different aggregates, from wrinkle and belt to fiber with the change of spacers and solvents. Spectral studies indicated that there existed different H-bond formations between imide groups and assembly modes, depending on the substituent spacers in molecular skeletons. The present work may give some insight into the design and character of new organogelators and soft materials with special molecular structures.

  15. Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate.

    PubMed Central

    Kokubu, F; Litman, R; Shamblott, M J; Hinds, K; Litman, G W

    1988-01-01

    The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH-D1-D2-JH-CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH-JH-CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH-CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH-D1-D2-JH-CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N-type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T-cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene. Images PMID:3145194

  16. Gene expression in the rodent brain is associated with its regional connectivity.

    PubMed

    Wolf, Lior; Goldberg, Chen; Manor, Nathan; Sharan, Roded; Ruppin, Eytan

    2011-05-01

    The putative link between gene expression of brain regions and their neural connectivity patterns is a fundamental question in neuroscience. Here this question is addressed in the first large scale study of a prototypical mammalian rodent brain, using a combination of rat brain regional connectivity data with gene expression of the mouse brain. Remarkably, even though this study uses data from two different rodent species (due to the data limitations), we still find that the connectivity of the majority of brain regions is highly predictable from their gene expression levels-the outgoing (incoming) connectivity is successfully predicted for 73% (56%) of brain regions, with an overall fairly marked accuracy level of 0.79 (0.83). Many genes are found to play a part in predicting both the incoming and outgoing connectivity (241 out of the 500 top selected genes, p-value<1e-5). Reassuringly, the genes previously known from the literature to be involved in axon guidance do carry significant information about regional brain connectivity. Surveying the genes known to be associated with the pathogenesis of several brain disorders, we find that those associated with schizophrenia, autism and attention deficit disorder are the most highly enriched in the connectivity-related genes identified here. Finally, we find that the profile of functional annotation groups that are associated with regional connectivity in the rodent is significantly correlated with the annotation profile of genes previously found to determine neural connectivity in C. elegans (Pearson correlation of 0.24, p<1e-6 for the outgoing connections and 0.27, p<1e-5 for the incoming). Overall, the association between connectivity and gene expression in a specific extant rodent species' brain is likely to be even stronger than found here, given the limitations of current data.

  17. The spacer arm length in cell-penetrating peptides influences chitosan/siRNA nanoparticle delivery for pulmonary inflammation treatment.

    PubMed

    Jeong, Eun Ju; Choi, Moonhwan; Lee, Jangwook; Rhim, Taiyoun; Lee, Kuen Yong

    2015-12-21

    Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of R9Gn-chitosan/siRNA nanoparticles were investigated in vitro. Increasing the spacing arm length did not significantly affect the complex formation between R9Gn-chitosan and siRNA. However, R9G10-chitosan was much more effective in delivering genes both in vitro and in vivo compared with non-modified chitosan (without the peptide) and R9-chitosan (without the spacer arm). Chitosan derivatives modified with oligoarginine containing a spacer arm can be considered as potential delivery vehicles for various genes.

  18. How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.

    PubMed

    Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

    2017-09-04

    CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes(1-5). Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer(6-9). Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical(7-9). The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle(6,10). The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3'-OH of each 3'-overhang serves as an attacking nucleophile during integration(11-14). Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array(1,7,10,15), guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat(7,15-20). Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)(21,22). In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro(18); other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis(17,23). Focusing on the Enterococcus faecalis Type II-A system(24), here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3'-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3'-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework

  19. Genetic diversity and molecular evolution of Naga King Chili inferred from internal transcribed spacer sequence of nuclear ribosomal DNA.

    PubMed

    Kehie, Mechuselie; Kumaria, Suman; Devi, Khumuckcham Sangeeta; Tandon, Pramod

    2016-02-01

    Sequences of the Internal Transcribed Spacer (ITS1-5.8S-ITS2) of nuclear ribosomal DNAs were explored to study the genetic diversity and molecular evolution of Naga King Chili. Our study indicated the occurrence of nucleotide polymorphism and haplotypic diversity in the ITS regions. The present study demonstrated that the variability of ITS1 with respect to nucleotide diversity and sequence polymorphism exceeded that of ITS2. Sequence analysis of 5.8S gene revealed a much conserved region in all the accessions of Naga King Chili. However, strong phylogenetic information of this species is the distinct 13 bp deletion in the 5.8S gene which discriminated Naga King Chili from the rest of the Capsicum sp. Neutrality test results implied a neutral variation, and population seems to be evolving at drift-mutation equilibrium and free from directed selection pressure. Furthermore, mismatch analysis showed multimodal curve indicating a demographic equilibrium. Phylogenetic relationships revealed by Median Joining Network (MJN) analysis denoted a clear discrimination of Naga King Chili from its closest sister species (Capsicum chinense and Capsicum frutescens). The absence of star-like network of haplotypes suggested an ancient population expansion of this chili.

  20. Assignment of genes to regions of mouse chromosomes.

    PubMed Central

    Eicher, E M; Washburn, L L

    1978-01-01

    A genetic mapping procedure, called the duplication-deficiency method, is described. This method permits the genetic location of a translocation to be determined within a linkage group without the use of recombination. By utilizing the duplication-deficiency method to define the genetic breakpoints for a series of translocations involving a given chromosome and integrating this information with their cytological breakpoints, obtained by Giemsa banding, a genetic map of the chromosomes is constructed whereby groups of loci are assigned to banded regions. Duplication-deficiency mapping and Giemsa banding analysis of the T(X;7)1Ct and T(7;19)145H translocations together with information from the c25H deletion have permitted mouse chromosome 7 to be divided into six and chromosome 19 into two definable genetic regions. Images PMID:273256

  1. Gene transfer to muscle from the isolated regional circulation.

    PubMed

    Petrov, Mihail; Malik, Alock; Mead, Andrew; Bridges, Charles R; Stedman, Hansell H

    2011-01-01

    Vector transport across the endothelium has long been regarded as one of the central "bottlenecks" in gene therapy research, especially as it pertains to the muscular dystrophies where the target tissue approaches half of the total body mass. Clinical studies of gene therapy for hemophilia B revealed the limitations of the intramuscular route, compelling an aggressive approach to the study of scale-independent circulatory means of vector delivery. The apparent permeability of the microvasculature in small animals suggests that gravitational and/or inertial effects on the circulation require progressive restriction of fluid and solute flow across the capillary wall with increasing body size. To overcome this physiological restriction, we initially used a combined surgical and pharmacological approach to temporarily alter permeability within the isolated pelvic limb. Although this was successful, new information about the cell and molecular biology of histamine-induced changes in microvascular permeability suggested an alternative approach, which substituted pressure-induced transvenular extravasation. Here we outline the details of our surgical approaches in the rat. We also discuss the modifications that are appropriate for the dog.

  2. Gene flow and selection interact to promote adaptive divergence in regions of low recombination.

    PubMed

    Samuk, Kieran; Owens, Gregory L; Delmore, Kira E; Miller, Sara E; Rennison, Diana J; Schluter, Dolph

    2017-09-01

    Adaptation to new environments often occurs in the face of gene flow. Under these conditions, gene flow and recombination can impede adaptation by breaking down linkage disequilibrium between locally adapted alleles. Theory predicts that this decay can be halted or slowed if adaptive alleles are tightly linked in regions of low recombination, potentially favouring divergence and adaptive evolution in these regions over others. Here, we compiled a global genomic data set of over 1,300 individual threespine stickleback from 52 populations and compared the tendency for adaptive alleles to occur in regions of low recombination between populations that diverged with or without gene flow. In support of theory, we found that putatively adaptive alleles (FST and dXY outliers) tend to occur more often in regions of low recombination in populations where divergent selection and gene flow have jointly occurred. This result remained significant when we employed different genomic window sizes, controlled for the effects of mutation rate and gene density, controlled for overall genetic differentiation, varied the genetic map used to estimate recombination and used a continuous (rather than discrete) measure of geographic distance as proxy for gene flow/shared ancestry. We argue that our study provides the first statistical evidence that the interaction of gene flow and selection biases divergence toward regions of low recombination. © 2017 John Wiley & Sons Ltd.

  3. Molecular Identification of Two Strains of Phellinus sp. by Internal Transcribed Spacer Sequence Analysis

    PubMed Central

    2011-01-01

    Two species of cultivated Phellinus sp. were identified as P. baumii by internal transcribed spacer (ITS) sequence analysis. The fruit bodies of the examined strains were similar to those of naturally occurring strains, having a bracket-like form, yellow-to-orange color, and poroid hymenial surfaces. The DNA sequences of ITS region of both strains showed a homology of 99% with ITS1 to ITS2 sequences of P. (Inonotus) baumii strain PB0806. PMID:22783119

  4. Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates.

    PubMed

    Chang, Perng-Kuang; Horn, Bruce W; Dorner, Joe W

    2005-11-01

    Aspergillus flavus populations are genetically diverse. Isolates that produce either, neither, or both aflatoxins and cyclopiazonic acid (CPA) are present in the field. We investigated defects in the aflatoxin gene cluster in 38 nonaflatoxigenic A. flavus isolates collected from southern United States. PCR assays using aflatoxin-gene-specific primers grouped these isolates into eight (A-H) deletion patterns. Patterns C, E, G, and H, which contain 40 kb deletions, were examined for their sequence breakpoints. Pattern C has one breakpoint in the cypA 3' untranslated region (UTR) and another in the verA coding region. Pattern E has a breakpoint in the amdA coding region and another in the ver1 5'UTR. Pattern G contains a deletion identical to the one found in pattern C and has another deletion that extends from the cypA coding region to one end of the chromosome as suggested by the presence of telomeric sequence repeats, CCCTAATGTTGA. Pattern H has a deletion of the entire aflatoxin gene cluster from the hexA coding region in the sugar utilization gene cluster to the telomeric region. Thus, deletions in the aflatoxin gene cluster among A. flavus isolates are not rare, and the patterns appear to be diverse. Genetic drift may be a driving force that is responsible for the loss of the entire aflatoxin gene cluster in nonaflatoxigenic A. flavus isolates when aflatoxins have lost their adaptive value in nature.

  5. Histone and Ribosomal RNA Repetitive Gene Clusters of the Boll Weevil are Linked in a Tandem Array

    USDA-ARS?s Scientific Manuscript database

    Histones are the major protein component of chromatin structure. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and ...

  6. Gene Expression in the Hippocampus: Regionally Specific Effects of Aging and Caloric Restriction

    PubMed Central

    Zeier, Zane; Madorsky, Irina; Xu, Ying; Ogle, William O.; Notterpek, Lucia; Foster, Thomas C.

    2010-01-01

    We measured changes in gene expression, induced by aging and caloric restriction (CR), in three hippocampal subregions. When analysis included all regions, aging was associated with expression of genes linked to mitochondrial dysfunction, inflammation, and stress responses, and in some cases, expression was reversed by CR. An age-related increase in ubiquintination was observed, including increased expression of ubiquitin conjugating enzyme genes and cytosolic ubiquitin immunoreactivity. CR decreased cytosolic ubiquitin and upregulated deubiquitinating genes. Region specific analyses indicated that CA1 was more susceptible to aging stress, exhibiting a greater number of altered genes relative to CA3 and the dentate gyrus (DG), and an enrichment of genes related to the immune response and apoptosis. CA3 and the DG were more responsive to CR, exhibiting marked changes in the total number of genes across diet conditions, reversal of age-related changes in p53 signaling, glucocorticoid receptor signaling, and enrichment of genes related to cell survival and neurotrophic signaling. Finally, CR differentially influenced genes for synaptic plasticity in CA1 and CA3. It is concluded that regional disparity in response to aging and CR relates to differences in vulnerability to stressors, the availability of neurotrophic, and cell survival mechanisms, and differences in cell function. PMID:21055414

  7. Pervasive generation of oppositely oriented spacers during CRISPR adaptation.

    PubMed

    Shmakov, Sergey; Savitskaya, Ekaterina; Semenova, Ekaterina; Logacheva, Maria D; Datsenko, Kirill A; Severinov, Konstantin

    2014-05-01

    During the process of prokaryotic CRISPR adaptation, a copy of a segment of foreign deoxyribonucleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a protospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (crRNA) transcribed from expanded CRISPR cassette can protect a prokaryotic cell from virus infection or plasmid transformation and conjugation. We show that in Escherichia coli, a vast majority of plasmid protospacers generate spacers integrated in CRISPR cassette in two opposing orientations, leading to frequent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adaptation. When a protospacer contains a spacer acquisition motif AAG, spacer orientation that generates functional protective crRNA is strongly preferred. All other protospacers give rise to spacers oriented in both ways at comparable frequencies. This phenomenon increases the repertoire of available spacers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation.

  8. Advanced hole patterning technology using soft spacer materials (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Park, Jong Keun; Hustad, Phillip D.; Aqad, Emad; Valeri, David; Wagner, Mike D.; Li, Mingqi

    2017-03-01

    A continuing goal in integrated circuit industry is to increase density of features within patterned masks. One pathway being used by the device manufacturers for patterning beyond the 80nm pitch limitation of 193 immersion lithography is the self-aligned spacer double patterning (SADP). Two orthogonal line space patterns with subsequent SADP can be used for contact holes multiplication. However, a combination of two immersion exposures, two spacer deposition processes, and two etch processes to reach the desired dimensions makes this process expensive and complicated. One alternative technique for contact hole multiplication is the use of an array of pillar patterns. Pillars, imaged with 193 immersion photolithography, can be uniformly deposited with spacer materials until a hole is formed in the center of 4 pillars. Selective removal of the pillar core gives a reversal of phases, a contact hole where there was once a pillar. However, the highly conformal nature of conventional spacer materials causes a problem with this application. The new holes, formed between 4 pillars, by this method have a tendency to be imperfect and not circular. To improve the contact hole circularity, this paper presents the use of both conventional spacer material and soft spacer materials. Application of soft spacer materials can be achieved by an existing coating track without additional cost burden to the device manufacturers.

  9. The 5S rDNA gene family in mollusks: characterization of transcriptional regulatory regions, prediction of secondary structures, and long-term evolution, with special attention to Mytilidae mussels.

    PubMed

    Vizoso, Miguel; Vierna, Joaquín; González-Tizón, Ana M; Martínez-Lage, Andrés

    2011-01-01

    Several reports on the characterization of 5S ribosomal DNA (5S rDNA) in various animal groups have been published to date, but there is a lack of studies analyzing this gene family in a much broader context. Here, we have studied 5S rDNA variation in several molluskan species, including bivalves, gastropods, and cephalopods. The degree of conservation of transcriptional regulatory regions was analyzed in these lineages, revealing a conserved TATA-like box in the upstream region. The evolution of the 120 bp coding region (5S) was also studied, suggesting the occurrence of paralogue groups in razor clams, clams, and cockles. In addition, 5S rDNA sequences from 11 species and 7 genus of Mytilidae Rafinesque, 1815 mussels were sampled and studied in detail. Four different 5S rDNA types, based on the nontranscribed spacer region were identified. The phylogenetic analyses performed within each type showed a between-species gene clustering pattern, suggesting ancestral polymorphism. Moreover, some putative pseudogenized 5S copies were also identified. Our report, together with previous studies that found high degree of intragenomic divergence in bivalve species, suggests that birth-and-death evolution may be the main force driving the evolution of 5S rDNA in these animals, even at the genus level.

  10. Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences.

    PubMed

    Kusumi, J; Tsumura, Y; Yoshimaru, H; Tachida, H

    2000-10-01

    Nucleotide sequences from four chloroplast genes, the matK, chlL, intergenic spacer (IGS) region between trnL and trnF, and an intron of trnL, were determined from all species of Taxodiaceae and five species of Cupressaceae sensu stricto (s.s.). Phylogenetic trees were constructed using the maximum parsimony and the neighbor-joining methods with Cunninghamia as an outgroup. These analyses provided greater resolution of relationships among genera and higher bootstrap supports for clades compared to previous analyses. Results indicate that Taiwania diverged first, and then Athrotaxis diverged from the remaining genera. Metasequoia, Sequoia, and Sequoiadendron form a clade. Taxodium and Glyptostrobus form a clade, which is the sister to Cryptomeria. Cupressaceae s.s. are derived from within Taxodiaceae, being the most closely related to the Cryptomeria/Taxodium/Glyptostrobus clade. These relationships are consistent with previous morphological groupings and the analyses of molecular data. In addition, we found acceleration of evolutionary rates in Cupressaceae s.s. Possible causes for the acceleration are discussed.

  11. Two 10-bp regions are critical for phytochrome regulation of a Lemna gibba Lhcb gene promoter.

    PubMed Central

    Kehoe, D M; Degenhardt, J; Winicov, I; Tobin, E M

    1994-01-01

    Two small regions of the promoter of an Lhcb gene encoding a light-harvesting chlorophyll a/b protein were identified as essential in conferring phytochrome responsiveness by using a transient expression assay. Initially, 5' deletion analysis of cabAB19, an Lhcb2 gene of Lemna, showed that sequences within the region from -174 to -104 relative to the start of transcription were necessary for phytochrome regulation. Internal deletion and substitution mutants were used to demonstrate that no additional phytochrome-responsive regions exist between -1600 and -174 in this promoter. A 171-bp fragment of the promoter extending from -239 to -69 was sufficient to impart phytochrome responsiveness to a minimal ubiquitin promoter that was not itself regulated by light. Specific binding of Lemna proteins to the region necessary for phytochrome responsiveness was demonstrated using in vitro polyacrylamide gel mobility shift assays and 1,10-phenanthroline copper ion footprinting. Further analysis of the region from -174 to -104 demonstrated that mutations in two separate 10-bp sequences, from -134 to -125 and from -114 to -105, could abolish phytochrome responsiveness; thus, there are two unique regions that are necessary for phytochrome regulation of this gene. One of these regions contains a CCAAT motif and the other a GATA motif. These motifs are conserved in the promoters of many Lhcb genes and may be important elements in the phytochrome responsiveness of this gene family. PMID:7919982

  12. Transport and deposition of pharmaceutical particles in three commercial spacer-MDI combinations.

    PubMed

    Yazdani, A; Normandie, M; Yousefi, M; Saidi, M S; Ahmadi, G

    2014-11-01

    Respiratory drug delivery has been under the research spotlight for the past few decades, mainly due to the high incidence of pulmonary diseases and the fact that this type of delivery offers the highest efficiency for treatment. Despite its invaluable benefits, there are some major drawbacks to respiratory drug delivery, the most important of which being poor delivery efficiency and relatively high drug deposition in undesirable regions, such as the mouth cavity. One way to improve the efficiency of respiratory drug delivery with metered-dose inhalers is placing a respiratory spacer between the inhaler exit and the mouth. It is argued that high drug deposition in the immediate airways of the respiratory system is strongly affected by relatively high initial momentum of pharmaceutical particles leaving the inhaler. A respiratory spacer, however, can provide an expansion region in which the initial momentum of particles can subside. As a result, particles enter the patient׳s oral cavity more gradually and are more likely to reach the desired regions. In this study, the effectiveness of using three commercial spacers paired with a commercial inhaler is examined through numerical investigation of fluid flow and particle transport phenomena. Particles ranging from 1 to 50 µm in diameter are tracked using a Lagrangian point of view and fluid flow fields are resolved using the LRN k-ω turbulence model. A novel particle injection method is introduced and is demonstrated to be able to adequately capture the effects of particle initial momentum. Lastly, a few design suggestions are made.

  13. Multilocus spacer analysis revealed highly homogeneous genetic background of Asian type of Borrelia miyamotoi.

    PubMed

    Mukhacheva, Tatyana A; Salikhova, Irina I; Kovalev, Sergey Y

    2015-04-01

    Borrelia miyamotoi, a member of the relapsing fever group borreliae, was first isolated in Japan and subsequently found in Ixodes ticks in North America, Europe and Russia. Currently, there are three types of B. miyamotoi: Asian or Siberian (transmitted mainly by Ixodes persulcatus), European (Ixodesricinus) and American (Ixodesscapularis and Ixodespacificus). Despite the great genetic distances between B. miyamotoi types, isolates within a type are characterised by an extremely low genetic variability. In particular, strains of B. miyamotoi of Asian type, isolated in Russia from the Baltic sea to the Far East, have been shown to be identical based on the analysis of several conventional genetic markers, such as 16S rRNA, flagellin, outer membrane protein p66 and glpQ genes. Thus, protein or rRNA - coding genes were shown not to be informative enough in studying genetic diversity of B. miyamotoi within a type. In the present paper, we have attempted to design a new multilocus technique based on eight non-coding intergenic spacers (3686bp in total) and have applied it to the analysis of intra-type genetic variability of В. miyamotoi detected in different regions of Russia and from two tick species, I. persulcatus and Ixodespavlovskyi. However, even though potentially the most variable loci were selected, no genetic variability between studied DNA samples was found, except for one nucleotide substitution in two of them. The sequences obtained were identical to those of the reference strain FR64b. Analysis of the data obtained with the GenBank sequences indicates a highly homogeneous genetic background of B. miyamotoi from the Baltic Sea to the Japanese Islands. In this paper, a hypothesis of clonal expansion of B. miyamotoi is discussed, as well as possible mechanisms for the rapid dissemination of one B. miyamotoi clone over large distances.

  14. Analysis of Mammalian rDNA Internal Transcribed Spacers

    PubMed Central

    Coleman, Annette W.

    2013-01-01

    Nuclear rDNA Internal Transcribed Spacers, ITS1 and ITS2, are widely used for eukaryote phylogenetic studies from the ordinal level to the species level, and there is even a database for ITS2 sequences. However, ITS regions have been ignored in mammalian phylogenetic studies, and only a few rodent and ape sequences are represented in GenBank. The reasons for this dearth, and the remedies, are described here. We have recovered these sequences, mostly >1 kb in length, for 36 mammalian species. Sequence alignment and transcript folding comparisons reveal the rRNA transcript secondary structure. Mammalian ITS regions, though quite long, still fold into the recognizable secondary structure of other eukaryotes. The ITS2 in particular bears the four standard helix loops, and loops II and III have the hallmark characters universal to eukaryotes. Both sequence and insertions/deletions of transcript secondary structure helices observed here support the four superorder taxonomy of Placentalia. On the family level, major unique indels, neatly excising entire helices, will be useful when additional species are represented, resulting in significant further understanding of the details of mammalian evolutionary history. Furthermore, the identification of a highly conserved element of ITS1 common to warm-blooded vertebrates may aid in deciphering the complex mechanism of RNA transcript processing. This is the last major group of terrestrial vertebrates for which rRNA ITS secondary structure has been resolved. PMID:24260162

  15. Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation.

    PubMed

    Botta, A; Lindsay, E A; Jurecic, V; Baldini, A

    1997-12-01

    We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies.

  16. Preclinical Evaluation of Bioabsorbable Polyglycolic Acid Spacer for Particle Therapy

    SciTech Connect

    Akasaka, Hiroaki; Sasaki, Ryohei; Miyawaki, Daisuke; Mukumoto, Naritoshi; Sulaiman, Nor Shazrina Binti; Nagata, Masaaki; Yamada, Shigeru; Murakami, Masao; Demizu, Yusuke; Fukumoto, Takumi

    2014-12-01

    Purpose: To evaluate the efficacy and safety of a polyglycolic acid (PGA) spacer through physical and animal experiments. Methods and Materials: The spacer was produced with surgical suture material made of PGA, forming a 3-dimensional nonwoven fabric. For evaluation or physical experiments, 150-MeV proton or 320-MeV carbon-ion beams were used to generate 60-mm width of spread-out Bragg peak. For animal experiments, the abdomens of C57BL/6 mice, with or without the inserted PGA spacers, were irradiated with 20 Gy of carbon-ion beam (290 MeV) using the spread-out Bragg peak. Body weight changes over time were scored, and radiation damage to the intestine was investigated using hematoxylin and eosin stain. Blood samples were also evaluated 24 days after the irradiation. Long-term thickness retention and safety were evaluated using crab-eating macaques. Results: No chemical or structural changes after 100 Gy of proton or carbon-ion irradiation were observed in the PGA spacer. Water equivalency of the PGA spacer was equal to the water thickness under wet condition. During 24 days' observation after 20 Gy of carbon-ion irradiation, the body weights of mice with the PGA spacer were relatively unchanged, whereas significant weight loss was observed in those mice without the PGA spacer (P<.05). In mice with the PGA spacer, villus and crypt structure were preserved after irradiation. No inflammatory reactions or liver or renal dysfunctions due to placement of the PGA spacer were observed. In the abdomen of crab-eating macaques, thickness of the PGA spacer was maintained 8 weeks after placement. Conclusions: The absorbable PGA spacer had water-equivalent, bio-compatible, and thickness-retaining properties. Although further evaluation is warranted in a clinical setting, the PGA spacer may be effective to stop proton or carbon-ion beams and to separate normal tissues from the radiation field.

  17. Inhaler spacer devices to treat asthma in children.

    PubMed

    Watson, Paul

    Drawing on literature searches and professional experience, this article discusses the treatment of asthma with pressurised metered dose inhalers (pMDIs). It demonstrates the need for pMDIs, and presents the health and cost benefits of using a pMDI through a spacer device. Through the review and evaluation of studies, it demonstrates the importance of correct asthma management and the use of spacers. Although there are many types of spacer, and patients often have less than optimal technique, there is evidence to support the overall benefits of use against non-use.

  18. GeneRegionScan: a Bioconductor package for probe-level analysis of specific, small regions of the genome.

    PubMed

    Folkersen, Lasse; Diez, Diego; Wheelock, Craig E; Haeggström, Jesper Z; Goto, Susumu; Eriksson, Per; Gabrielsen, Anders

    2009-08-01

    Whole-genome microarrays allow us to interrogate the entire transcriptome of a cell. Affymetrix microarrays are constructed using several probes that match to different regions of a gene and a summarization step reduces this complexity into a single value, representing the expression level of the gene or the expression level of an exon in the case of exon arrays. However, this simplification eliminates information that might be useful when focusing on specific genes of interest. To address these limitations, we present a software package for the R platform that allows detailed analysis of expression at the probe level. The package matches the probe sequences against a target gene sequence (either mRNA or DNA) and shows the expression levels of each probe along the gene. It also features functions to fit a linear regression based on several genetic models that enables study of the relationship between gene expression and genotype. The software is implemented as a platform-independent R package available through the Bioconductor repository at http://www.bioconductor.org/. It is licensed as GPL 2.0. Supplementary data are available at Bioinformatics online.

  19. Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design

    PubMed Central

    Zhang, Luchen; Li, Zhenbo; Sun, Fangli; Xu, Yuhong; Du, Zixiu

    2016-01-01

    A safe and efficient quaternary gene delivery system (named Q-complexes) was constructed based on self-assembly of molecules through noncovalent bonds. This system was formulated through the cooperation and competing interactions of cationic liposomes, multifunctional peptides, and DNA, followed by coating hyaluronic acid on the surface of the ternary complexes. The multifunctional peptide was composed of two functional domains: penetrating hepatic tumor-targeted cell moiety (KRPTMRFRYTWNPMK) and a wrapping gene sequence (polyarginine 16). The effect of spacer insertion between the two domains of multifunctional peptide on the intracellular transfection of Q-complexes was further studied. Experimental results showed that the formulations assembled with various peptides in the spacer elements possessed different intercellular pathways and transfection efficiencies. The Q-complexes containing peptide in the absence of spacer element (Pa) showed the highest gene expression among all samples. The Q-complexes containing peptides with a noncleavable spacer GA (Pc) had no ability of intracellular nucleic acid delivery, whereas those with a cleavable spacer RVRR (Pd) showed moderate transfection activity. These results demonstrated that the different spacers inserted in the multifunctional peptide played an important role in in vitro DNA transfection efficiency. Atomic force microscopy images showed that the morphologies of ternary complexes (LPcD) and Q-complexes (HLcPD) were crystal lamellas, whereas those of other nanocomplexes were spheres. Circular dichroism showed the changed configuration of peptide with spacer GA in nanocomplexes compared with that of its free state, whereas the Pa configuration without spacer in nanocomplexes was consistent with that of its free state. The present study contributed to the structural understanding of Q-complexes, and further effective modification is in progress. PMID:27920533

  20. Cloning and characterization of the 5'-flanking region of the Ehox gene

    SciTech Connect

    Lee, Woon Kyu . E-mail: wklee@yumc.yonsei.ac.kr; Kim, Yong-Man; Malik, Nasir; Ma Chang; Westphal, Heiner

    2006-03-03

    The paired-like homeobox-containing gene Ehox plays a role in embryonic stem cell differentiation and is highly expressed in the developing placenta and thymus. To understand the mechanisms of regulation of Ehox gene expression, the 5'-flanking region of the Ehox gene was isolated from a mouse BAC library. 5'-RACE analysis revealed a single transcriptional start site 130 nucleotides upstream of the translation initiation codon. Transient transfection with a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the nt -84 to -68 region contained a positive cis-acting element for efficient expression of the Ehox gene. Mutational analysis of this region and oligonucleotide competition in the electrophoretic mobility shift assay revealed the presence of a CCAAT box, which is a target for transcription nuclear factor Y (NFY). NFY is essential for positive gene regulation. No tissue-specific enhancer was identified in the 1.9-kb 5'-flanking region of the Ehox gene. Ehox is expressed during the early stages of embryo development, specifically in Brain at 9.5 dpc, as well as during the late stages of embryo development. These results suggest that NFY is an essential regulatory factor for Ehox transcriptional activity, which is important for the post-implantation stage of the developing embryo.

  1. Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor.

    PubMed

    Ryan, J J; McReynolds, L J; Keegan, A; Wang, L H; Garfein, E; Rothman, P; Nelms, K; Paul, W E

    1996-02-01

    IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557-657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557-657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R.

  2. Methylation of coding region alone inhibits gene expression in plant protoplasts.

    PubMed Central

    Hohn, T; Corsten, S; Rieke, S; Müller, M; Rothnie, H

    1996-01-01

    Derivatives of the cauliflower mosaic virus 35S promoter lacking CG and CNG methylation targets were constructed and used to direct transcription of reporter gene constructs in transiently transformed protoplasts. Such methylation-target-free (MTF) promoters, although weaker than the 35S promoter, retain significant activity despite mutation of the as-1 element. The effect of methylation on gene expression in MTF- and 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was markedly reduced when cytosine residues in CG dinucleotides were methylated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, revealed that methylation of the coding region alone has a negative effect on reporter gene expression. Methylation nearer the 5' end of the coding region was more inhibitory, consistent with inhibition of transcription elongation. Images Fig. 5 PMID:8710871

  3. Circuit-wide transcriptional profiling reveals brain region-specific gene networks regulating depression susceptibility

    PubMed Central

    Bagot, Rosemary C.; Cates, Hannah M.; Purushothaman, Immanuel; Lorsch, Zachary S.; Walker, Deena M.; Wang, Junshi; Huang, Xiaojie; Schlüter, Oliver M.; Maze, Ian; Peña, Catherine J.; Heller, Elizabeth A.; Issler, Orna; Wang, Minghui; Song, Won-min; Stein, Jason. L.; Liu, Xiaochuan; Doyle, Marie A.; Scobie, Kimberly N.; Sun, Hao Sheng; Neve, Rachael L.; Geschwind, Daniel; Dong, Yan; Shen, Li; Zhang, Bin; Nestler, Eric J.

    2016-01-01

    Summary Depression is a complex, heterogeneous disorder and a leading contributor to the global burden of disease. Most previous research has focused on individual brain regions and genes contributing to depression. However, emerging evidence in humans and animal models suggests that dysregulated circuit function and gene expression across multiple brain regions drive depressive phenotypes. Here we performed RNA-sequencing on 4 brain regions from control animals and those susceptible or resilient to chronic social defeat stress at multiple time points. We employed an integrative network biology approach to identify transcriptional networks and key driver genes that regulate susceptibility to depressive-like symptoms. Further, we validated in vivo several key drivers and their associated transcriptional networks that regulate depression susceptibility and confirmed their functional significance at the levels of gene transcription, synaptic regulation and behavior. Our study reveals novel transcriptional networks that control stress susceptibility and offers fundamentally new leads for antidepressant drug discovery. PMID:27181059

  4. Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo.

    PubMed Central

    Zwingman, T; Erickson, R P; Boyer, T; Ao, A

    1993-01-01

    We have confirmed the faster growth of male preimplantation mouse embryos. We have also studied the transcription of Y chromosomal genes postulated to have a role in sex determination, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. We find that two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. We also show that the anti-Müllerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation. Images PMID:8430091

  5. Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo.

    PubMed

    Zwingman, T; Erickson, R P; Boyer, T; Ao, A

    1993-02-01

    We have confirmed the faster growth of male preimplantation mouse embryos. We have also studied the transcription of Y chromosomal genes postulated to have a role in sex determination, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. We find that two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. We also show that the anti-Müllerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation.

  6. Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt.

    PubMed

    Gjerde, Bjørn; Hilali, Mosaad; Mawgood, Sahar Abdel

    2015-09-01

    A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15

  7. Analysis of the genetic diversity of the Plasmodium falciparum multidrug resistance gene 5' upstream region.

    PubMed

    Myrick, Alissa; Sarr, Ousmane; Dieng, Therese; Ndir, Omar; Mboup, Souleymane; Wirth, Dyann F

    2005-02-01

    Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal. A comparison of six geographically diverse laboratory strains showed that the 1.94-kb 5'-untranslated region is highly monomorphic, with a total of four unique single nucleotide polymorphisms (SNPs) being identified. All of the mutations were localized to a 462-basepair region proximal to the transcription start point. Analysis of this region in field isolates shows the prevalence of one SNP throughout the entire population of parasites, irrespective of drug resistance status. The SNP frequency of the pfmdr1 upstream region is lower than that found in the noncoding region of other genes.

  8. Methylation status of the promoter region of the human frizzled 9 gene in acute myeloid leukemia.

    PubMed

    Zhang, Yingjie; Jiang, Qi; Kong, Xiaolin; Yang, Lili; Hu, Wanzhen; Lv, Chengfang; Li, Yinghua

    2016-08-01

    The FZD9 gene is located at chromosome 7q11.23, and has been indicated to be a tumor suppressor gene. The present study examined the involvement of FZD9 promoter methylation in the downregulation of FZD9 expression in leukemia cells. The expression of the FZD9 gene was absent in various leukemic cell lines, while it was restored following treatment with DNA demethylating agent 5‑aza‑2'‑deoxycytidine. Bisulfite sequencing analysis of the FZD9 promoter region showed that it was partially methylated in cell lines in which FZD9 gene was not expressed. Thus, DNA methylation in the promoter region may lead to inactivation of the FZD9 gene, which may represent and aberration associated with leukemia, since DNA was not methylated in normal peripheral blood mononuclear cells. Methylation‑specific polymerase chain reaction analysis revealed that the promoter region of the FZD9 gene was frequently methylated in primary or relapse acute myeloid leukemia (52.9%; excluding acute promyelocytic leukemia); however, methylation was infrequent in B‑cell acute lymphocytic leukemia (5.6%). In conclusion, the present study indicated that the methylation profile of the FZD9 gene corresponded to that of a candidate tumor‑suppressor gene in acute myeloid leukemia.

  9. Sequence and analysis of the human ABL gene, the BCR gene, and regions involved in the Philadelphia chromosomal translocation

    SciTech Connect

    Burian, D.; Clifton, S.W.; Crabtree, J.

    1995-05-01

    The complete human BCR gene (152j-141 nt) on chromosome 22 and greater than 80% of the human ABL gene (179-512 nt) on chromosome 9 have been sequenced from mapped cosmid and plasmid clones via a shotgun strategy. Because these two chromosomes are translocated with breakpoints within the BCR and ABL genes in Philadelphia chromosome-positive leukemias, knowledge of these sequences also might provide insight into the validity of various theories of chromosomal rearrangements. Comparison of these genes with their cDNA sequences reveal the positions of 23 BCR exons and putative alternative BCR first and second exons, as well as the common ABL exons 2-11, respectively. Additionally, these regions include the alternative ABL first exons 1b and 1a, a new gene 5` to the first ABL exon, and an open reading frame with homology to an EST within the BCR fourth intron. Further analysis reveals an Alu homology of 38.83 and 39.35% for the BCR and ABL genes, respectively, with other repeat elements present to a lesser extent. Four new Philadelphia chromosome translocation breakpoints from chronic myelogenous leukemia patients also were sequenced, and the positions of these and several other previously sequenced breakpoints now have been mapped precisely, although no consistent breakpoint features immediately were apparent. Comparative analysis of genomic sequences encompassing the murine homologues to the human ABL exons 1b and 1a, as well as regions encompassing the ABL exons 2 and 3, reveals that although there is a high degree of homology in their corresponding exons and promoter regions, these two vertebrate species show a striking lack of homology outside these regions. 122 refs., 5 figs., 4 tabs.

  10. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

    PubMed Central

    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.

    1995-01-01

    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher. PMID:8529116

  11. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide.

    PubMed

    Lowenstein, C J; Alley, E W; Raval, P; Snowman, A M; Snyder, S H; Russell, S W; Murphy, W J

    1993-10-15

    The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.

  12. Utility of indels for species-level identification of a biologically complex plant group: a study with intergenic spacer in Citrus.

    PubMed

    Mahadani, Pradosh; Ghosh, Sankar Kumar

    2014-11-01

    The Consortium of Barcode of Life plant working group proposed to use the defined portion of plastid genes rbcL and matK either singly or in combination as the standard DNA barcode for plants. But DNA barcode based identification of biologically complex plant groups are always a challenging task due to the occurrence of natural hybridization. Here, we examined the use of indels polymorphism in trnH-psbA and trnL-trnF sequences for rapid species identification of citrus. DNA from young leaves of selected citrus species were isolated and matK gene (~800 bp) and trnH-psbA spacer (~450 bp) of Chloroplast DNA was amplified for species level identification. The sequences within the group taxa of Citrus were aligned using the ClustalX program. With few obvious misalignments were corrected manually using the similarity criterion. We identified a 54 bp inverted repeat or palindrome sequence (27-80 regions) and 6 multi residues indel coding regions. Large inverted repeats in cpDNA provided authentication at the higher taxonomic levels. These diagnostics indel marker from trnH-psbA were successful in identifying different species (5 out of 7) within the studied Citrus except Citrus limon and Citrus medica. These two closely related species are distinguished through the 6 bp deletion in trnL-trnF. This study demonstrated that the indel polymorphism based approach easily characterizes the Citrus species and the same may be applied in other complex groups. Likewise other indels occurring intergenic spacer of chloroplast regions may be tested for rapid identification of other secondary citrus species.

  13. A 4.5-megabase YAC contig and physical map over the hemochromatosis gene region

    SciTech Connect

    Burt, M.J.; Smit, D.J.; Pyper, W.R.; Powell, L.W.; Jazwinska, E.C.

    1996-04-15

    We have constructed a yeast artificial chromosome (YAC) contig over the candidate hemochromatosis gene region. This contig comprises hemochromatosis gene region. This contig comprises 16 YACs from the CEPH, Washington University, and ICI YAC libraries and covers 4.5 Mb at 6p21.3-6p22. The complete contig has been restriction mapped, enabling the precise relationship between the YACs to be determined and the mapping of a total of 12 STSs. Nine of these are highly polymorphic STSs that are closely linked to hemochromatosis; this series includes D6S265 and D6S1260, which comprise the most proximal and distal markers linked to HC. This is the first YAC contig that spans the hemochromatosis candidate region, and it provides valuable resource material for the cloning of this and other genes in the region distal to the MHC class I complex. 33 refs., 1 fig., 1 tab.

  14. The mitochondrial genome of Paragyrodactylus variegatus (Platyhelminthes: Monogenea): differences in major non-coding region and gene order compared to Gyrodactylus.

    PubMed

    Ye, Fei; King, Stanley D; Cone, David K; You, Ping

    2014-08-17

    Paragyrodactylus Gvosdev and Martechov, 1953, a viviparous genus of ectoparasite within the Gyrodactylidae, contains three nominal species all of which infect Asian river loaches. The group is suspected to be a basal lineage within Gyrodactylus Nordmann, 1832 sensu lato although this remains unclear. Further molecular study, beyond characterization of the standard Internal Transcribed Spacer region, is needed to clarify the evolutionary relationships within the family and the placement of this genus. The mitochondrial genome of Paragyrodactylus variegatus You, King, Ye and Cone, 2014 was amplified in six parts from a single worm, sequenced using primer walking, annotated and analyzed using bioinformatic tools. The mitochondrial genome of P. variegatus is 14,517 bp, containing 12 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a major non-coding region (NCR). The overall A + T content of the mitochondrial genome is 76.3%, which is higher than all reported mitochondrial genomes of monogeneans. All of the 22 tRNAs have the typical cloverleaf secondary structure, except tRNACys, tRNASer1 and tRNASer2 that lack the dihydrouridine (DHU) arm. There are six domains (domain III is absent) and three domains in the inferred secondary structures of the large ribosomal subunit (rrnL) and small ribosomal subunit (rrnS), respectively. The NCR includes six 40 bp tandem repeat units and has the double identical poly-T stretches, stem-loop structure and some surrounding structure elements. The gene order (tRNAGln, tRNAMet and NCR) differs in arrangement compared to the mitochondrial genomes reported from Gyrodactylus spp. The Duplication and Random Loss Model and Recombination Model together are the most plausible explanations for the variation in gene order. Both morphological characters and characteristics of the mitochondrial genome support Paragyrodactylus as a distinct genus from Gyrodactylus. Considering their specific

  15. High sequence turnover in the regulatory regions of the developmental gene hunchback in insects.

    PubMed

    Hancock, J M; Shaw, P J; Bonneton, F; Dover, G A

    1999-02-01

    Extensive sequence analysis of the developmental gene hunchback and its 5' and 3' regulatory regions in Drosophila melanogaster, Drosophila virilis, Musca domestica, and Tribolium castaneum, using a variety of computer algorithms, reveals regions of high sequence simplicity probably generated by slippage-like mechanisms of turnover. No regions are entirely refractory to the action of slippage, although the density and composition of simple sequence motifs varies from region to region. Interestingly, the 5' and 3' flanking regions share short repetitive motifs despite their separation by the gene itself, and the motifs are different in composition from those in the exons and introns. Furthermore, there are high levels of conservation of motifs in equivalent orthologous regions. Detailed sequence analysis of the P2 promoter and DNA footprinting assays reveal that the number, orientation, sequence, spacing, and protein-binding affinities of the BICOID-binding sites varies between species and that the 'P2' promoter, the nanos response element in the 3' untranslated region, and several conserved boxes of sequence in the gene (e.g., the two zinc-finger regions) are surrounded by cryptically-simple-sequence DNA. We argue that high sequence turnover and genetic redundancy permit both the general maintenance of promoter functions through the establishment of coevolutionary (compensatory) changes in cis- and trans-acting genetic elements and, at the same time, the possibility of subtle changes in the regulation of hunchback in the different species.

  16. A brain region-specific predictive gene map for autism derived by profiling a reference gene set.

    PubMed

    Kumar, Ajay; Swanwick, Catherine Croft; Johnson, Nicole; Menashe, Idan; Basu, Saumyendra N; Bales, Michael E; Banerjee-Basu, Sharmila

    2011-01-01

    Molecular underpinnings of complex psychiatric disorders such as autism spectrum disorders (ASD) remain largely unresolved. Increasingly, structural variations in discrete chromosomal loci are implicated in ASD, expanding the search space for its disease etiology. We exploited the high genetic heterogeneity of ASD to derive a predictive map of candidate genes by an integrated bioinformatics approach. Using a reference set of 84 Rare and Syndromic candidate ASD genes (AutRef84), we built a composite reference profile based on both functional and expression analyses. First, we created a functional profile of AutRef84 by performing Gene Ontology (GO) enrichment analysis which encompassed three main areas: 1) neurogenesis/projection, 2) cell adhesion, and 3) ion channel activity. Second, we constructed an expression profile of AutRef84 by conducting DAVID analysis which found enrichment in brain regions critical for sensory information processing (olfactory bulb, occipital lobe), executive function (prefrontal cortex), and hormone secretion (pituitary). Disease specificity of this dual AutRef84 profile was demonstrated by comparative analysis with control, diabetes, and non-specific gene sets. We then screened the human genome with the dual AutRef84 profile to derive a set of 460 potential ASD candidate genes. Importantly, the power of our predictive gene map was demonstrated by capturing 18 existing ASD-associated genes which were not part of the AutRef84 input dataset. The remaining 442 genes are entirely novel putative ASD risk genes. Together, we used a composite ASD reference profile to generate a predictive map of novel ASD candidate genes which should be prioritized for future research.

  17. An integrative analysis of regional gene expression profiles in the human brain.

    PubMed

    Myers, Emma M; Bartlett, Christopher W; Machiraju, Raghu; Bohland, Jason W

    2015-02-01

    Studies of the brain's transcriptome have become prominent in recent years, resulting in an accumulation of datasets with somewhat distinct attributes. These datasets, which are often analyzed only in isolation, also are often collected with divergent goals, which are reflected in their sampling properties. While many researchers have been interested in sampling gene expression in one or a few brain areas in a large number of subjects, recent efforts from the Allen Institute for Brain Sciences and others have focused instead on dense neuroanatomical sampling, necessarily limiting the number of individual donor brains studied. The purpose of the present work is to develop methods that draw on the complementary strengths of these two types of datasets for study of the human brain, and to characterize the anatomical specificity of gene expression profiles and gene co-expression networks derived from human brains using different specific technologies. The approach is applied using two publicly accessible datasets: (1) the high anatomical resolution Allen Human Brain Atlas (AHBA, Hawrylycz et al., 2012) and (2) a relatively large sample size, but comparatively coarse neuroanatomical dataset described previously by Gibbs et al. (2010). We found a relatively high degree of correspondence in differentially expressed genes and regional gene expression profiles across the two datasets. Gene co-expression networks defined in individual brain regions were less congruent, but also showed modest anatomical specificity. Using gene modules derived from the Gibbs dataset and from curated gene lists, we demonstrated varying degrees of anatomical specificity based on two classes of methods, one focused on network modularity and the other focused on enrichment of expression levels. Two approaches to assessing the statistical significance of a gene set's modularity in a given brain region were studied, which provide complementary information about the anatomical specificity of a gene

  18. 14. TYPICAL WORK DECK SHOWING RING SPACERS, CABLE DRUMS AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. TYPICAL WORK DECK SHOWING RING SPACERS, CABLE DRUMS AND OTHER SPECIALIZED HARDWARE; VIEW TO SOUTH. - Cape Canaveral Air Station, Launch Complex 17, Facility 28416, East end of Lighthouse Road, Cape Canaveral, Brevard County, FL

  19. Technique for adapting a spacer for a custom impression tray.

    PubMed

    Kaur, Harsimran; Nanda, Aditi; Verma, Mahesh; Koli, Dheeraj

    2016-12-01

    A method of adapting a spacer for the custom trays used to make a definite impression for complete dentures is presented. The technique can be used under a variety of conditions and offers several advantages.

  20. Genetic Architecture of MAPT Gene Region in Parkinson Disease Subtypes.

    PubMed

    Pascale, Esterina; Di Battista, Maria Elena; Rubino, Alfonso; Purcaro, Carlo; Valente, Marcella; Fattapposta, Francesco; Ferraguti, Giampiero; Meco, Giuseppe

    2016-01-01

    The microtubule-associated protein tau (MAPT) region has been conceptualized as a model of the interaction between genetics and functional disease outcomes in neurodegenerative disorders, such as Parkinson disease (PD). Indeed, haplotype-specific differences in expression and alternative splicing of MAPT transcripts affect cellular functions at different levels, increasing susceptibility to a range of neurodegenerative processes. In order to evaluate a possible link between MAPT variants, PD risk and PD motor phenotype, we analyzed the genetic architecture of MAPT in a cohort of PD patients. We observed a statistically significant association between the H1 haplotype and PD risk (79.5 vs 69.5%; χ(2) = 9.9; OR, 1.7; 95% CI, 1.2-2.4; p = 0.002). The effect was more evident in non tremor dominant (TD) PD subjects (NTD-PD) (82 vs 69.5%; χ(2) = 13.6; OR, 2.03; 95% CI, 1.4-3; p = 0.0003), while no difference emerged between PD subgroup of tremor dominant patients (TD-PD) and control subjects. Examination of specific intra-H1 variations showed that the H1h subhaplotype was overrepresented in NTD-PD patients compared with controls (p = 0.007; OR, 2.9; 95% CI, 1.3-6.3). Although we cannot exclude that MAPT variation may be associated with ethnicity, our results may support the hypothesis that MAPT H1 clade and a specific H1 subhaplotype influence the risk of PD and modulate the clinical expression of the disease, including motor phenotype.

  1. Genetic Architecture of MAPT Gene Region in Parkinson Disease Subtypes

    PubMed Central

    Pascale, Esterina; Di Battista, Maria Elena; Rubino, Alfonso; Purcaro, Carlo; Valente, Marcella; Fattapposta, Francesco; Ferraguti, Giampiero; Meco, Giuseppe

    2016-01-01

    The microtubule-associated protein tau (MAPT) region has been conceptualized as a model of the interaction between genetics and functional disease outcomes in neurodegenerative disorders, such as Parkinson disease (PD). Indeed, haplotype-specific differences in expression and alternative splicing of MAPT transcripts affect cellular functions at different levels, increasing susceptibility to a range of neurodegenerative processes. In order to evaluate a possible link between MAPT variants, PD risk and PD motor phenotype, we analyzed the genetic architecture of MAPT in a cohort of PD patients. We observed a statistically significant association between the H1 haplotype and PD risk (79.5 vs 69.5%; χ2 = 9.9; OR, 1.7; 95% CI, 1.2–2.4; p = 0.002). The effect was more evident in non tremor dominant (TD) PD subjects (NTD-PD) (82 vs 69.5%; χ2 = 13.6; OR, 2.03; 95% CI, 1.4–3; p = 0.0003), while no difference emerged between PD subgroup of tremor dominant patients (TD-PD) and control subjects. Examination of specific intra-H1 variations showed that the H1h subhaplotype was overrepresented in NTD-PD patients compared with controls (p = 0.007; OR, 2.9; 95% CI, 1.3–6.3). Although we cannot exclude that MAPT variation may be associated with ethnicity, our results may support the hypothesis that MAPT H1 clade and a specific H1 subhaplotype influence the risk of PD and modulate the clinical expression of the disease, including motor phenotype. PMID:27147968

  2. Identification, characterization and phylogenic analysis of conserved genes within the p74 gene region of Choristoneura fumiferana granulovirus genome.

    PubMed

    Rashidan, Kianoush Khajeh; Nassoury, Nasha; Giannopoulos, Paresa N; Mauffette, Yves; Guertin, Claude

    2004-11-30

    The genes located within the p74 gene region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing an 8.9 kb BamHI restriction fragment on the ChfuGV genome. The global guanine-cytosine (GC) content of this region of the genome was 33.02%. This paper presents the ORFs within the p74 gene region along with their transcriptional orientations. This region contains a total of 15 open reading frames (ORFs). Among those, 8 ORFs were found to be homologues to the baculoviral ORFs: Cf-i-p , Cf-vi, Cf-vii, Cf-viii (ubiquitin), Cf-xi (pp31), Cf-xii (lef-11), Cf-xiii (sod) and Cf-xv-p (p74). To date, no specific function has been assigned to the ORFs: Cf-i, Cf-ii, Cf-iii, Cf-iv, Cf-v, Cf-vi, Cf-vii, Cf-ix and Cf-x. The most noticeable ORFs located in this region of the ChfuGV genome were ubiquitin, lef-11, sod, fibrillin and p74. The phylogenetic trees (constructed using conceptual products of major conserved ORFs) and gene arrangement in this region were used to further examine the classification of the members of the granulovirus genus. Comparative studies demonstrated that ChfuGV along with the Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), Adoxophyes orana granulovirus (AoGV) and Cryptophlebia leucotreta granulovirus (ClGV) share a high degree of amino acids sequence and gene arrangement preservation within the studied region. These results support a previous report, which classified a granuloviruses into 2 distinct groups: Group I: ChfuGV, CpGV, PhopGV and AoGV and Group II: Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The phylogenetic and gene arrangement studies also placed ClGV as a novel member of the Group I granuloviruses.

  3. Invariance of the magnetic behavior and AMI in ferromagnetic biphase films with distinct non-magnetic metallic spacers

    NASA Astrophysics Data System (ADS)

    Silva, E. F.; Gamino, M.; Andrade, A. M. H. de; Vázquez, M.; Correa, M. A.; Bohn, F.

    2017-02-01

    We investigate the quasi-static magnetic, magnetotransport, and dynamic magnetic properties in ferromagnetic biphase films with distinct non-magnetic metallic spacer layers. We observe that the nature of the non-magnetic metallic spacer material does not have significant influence on the overall biphase magnetic behavior, and, consequently, on the magnetotransport and dynamic magnetic responses. We focus on the magnetoimpedance effect and verify that the films present asymmetric magnetoimpedance effect. Moreover, we explore the possibility of tuning the linear region of the magnetoimpedance curves around zero magnetic field by varying the probe current frequency in order to achieve higher sensitivity values. The invariance of the magnetic behavior and the asymmetric magnetoimpedance effect in ferromagnetic biphase films with distinct non-magnetic metallic spacers place them as promising candidates for probe element and open possibilities to the development of lower-cost high sensitivity linear magnetic field sensor devices.

  4. Mosaic Tetracycline Resistance Genes and Their Flanking Regions in Bifidobacterium thermophilum and Lactobacillus johnsonii▿

    PubMed Central

    van Hoek, Angela H. A. M.; Mayrhofer, Sigrid; Domig, Konrad J.; Flórez, Ana B.; Ammor, Mohammed S.; Mayo, Baltasar; Aarts, Henk J. M.

    2008-01-01

    For the first time, mosaic tetracycline resistance genes were identified in Lactobacillus johnsonii and in Bifidobacterium thermophilum strains. The L. johnsonii strain investigated contains a complex hybrid gene, tet(O/W/32/O/W/O), whereas the five bifidobacterial strains possess two different mosaic tet genes: i.e., tet(W/32/O) and tet(O/W). As reported by others, the crossover points of the mosaic tet gene segments were found at similar positions within the genes, suggesting a hot spot for recombination. Analysis of the sequences flanking these genes revealed that the upstream part corresponds to the 5′ end of the mosaic open reading frame. In contrast, the downstream region was shown to be more variable. Surprisingly, in one of the B. thermophilum strains a third tet determinant was identified, coding for the efflux pump Tet(L). PMID:17967912

  5. Improvement of pressurised aerosol deposition with Nebuhaler spacer device.

    PubMed Central

    Newman, S P; Millar, A B; Lennard-Jones, T R; Morén, F; Clarke, S W

    1984-01-01

    The effect on aerosol deposition from a pressurised metered dose inhaler of a 750 cm3 spacer device with a one way inhalation valve (Nebuhaler, Astra Pharmaceuticals) was assessed by means of an in vivo radiotracer technique. Nine patients with obstructive lung disease took part in the study. The pattern of deposition associated with use of a metered dose inhaler alone was compared with that achieved with the spacer used both for inhalation of single puffs of aerosol and for inhalation of four puffs actuated in rapid succession and then inhaled simultaneously. On each occasion there was a delay of 1 s between aerosol release and inhalation, simulating poor inhaler technique. With the metered dose inhaler alone, a mean (SEM) 8.7 (1.8)% of the dose reached the lungs and 80.9 (1.9)% was deposited in the oropharynx. With single puffs from the spacer 20.9 (1.6)% of the dose (p less than 0.01) reached the lungs, only 16.5 (2.3)% (p less than 0.01) was deposited in the oropharynx, and 55.8 (3.1)% was retained within the spacer itself. With four puffs from the spacer 15.2 (1.5)% reached the lungs (p = 0.02 compared with the metered dose inhaler alone, p less than 0.01 compared with single puffs from the spacer), 11.4 (1.2)% was deposited in the oropharynx, and 67.5 (1.8)% in the device itself. It is concluded that the spacer device gives lung deposition of metered dose aerosols comparable to or greater than a correctly used inhaler and oropharyngeal deposition is greatly reduced. The spacer should be used preferably for the inhalation of single puffs of aerosol but may also be used for the inhalation of up to four puffs actuated in rapid succession and then inhaled simultaneously. Images PMID:6440305

  6. Assessing the Value of DNA Barcodes and Other Priority Gene Regions for Molecular Phylogenetics of Lepidoptera

    PubMed Central

    Wilson, John James

    2010-01-01

    Background Despite apparently abundant amounts of observable variation and species diversity, the order Lepidoptera exhibits a morphological homogeneity that has provided only a limited number of taxonomic characters and led to widespread use of nucleotides for inferring relationships. This study aims to characterize and develop methods to quantify the value of priority gene regions designated for Lepidoptera molecular systematics. In particular, I assess how the DNA barcode segment of the mitochondrial COI gene performs across a broad temporal range given its number one position of priority, most sequenced status, and the conflicting opinions on its phylogenetic performance. Methodology/Principal Findings Gene regions commonly sequenced for Lepidoptera phylogenetics were scored using multiple measures across three categories: practicality, which includes universality of primers and sequence quality; phylogenetic utility; and phylogenetic signal. I found that alternative measures within a category often appeared correlated, but high scores in one category did not necessarily translate into high scores in another. The DNA barcode was easier to sequence than other genes, and had high scores for utility but low signal above the genus level. Conclusions/Significance Given limited financial resources and time constraints, careful selection of gene regions for molecular phylogenetics is crucial to avoid wasted effort producing partially informative data. This study introduces an approach to assessing the value of gene regions prior to the initiation of new studies and presents empirical results to help guide future selections. PMID:20479871

  7. Association study of proposed candidate genes/regions in a population of Spanish asthmatics.

    PubMed

    Soriano, J B; de Cid, R; Estivill, X; Antó, J M; Sunyer, J; Otero, D; Roca, J; Rodríguez-Roisín, R; Morell, F; Rodrigo, M J; Ercilla, G; Beaty, T H; Lázaro, C

    2000-01-01

    A number of genes/regions have recently been reported to be linked to asthma or its related phenotypes (i.e. atopy and bronchial hyperresponsiveness), by genetic linkage and allele-sharing methods. We have performed a case-control study comparing the allelic distribution of nine microsatellite markers and two genetic variants in a group of patients attended at emergency room departments because of an acute attack of asthma with respect to an external healthy population of controls. A total of 146 asthmatic subjects and 50 population controls from Barcelona, Spain, were genotyped for nine microsatellite markers from some asthma/atopy candidate genes/regions: the beta-subunit of the high-affinity IgE receptor (Fc epsilonRI-beta) located on chromosome 11; the 5q31-32 candidate region; the T-cell receptor genes, TCR-alpha on chromosome 14 and TCR-beta on chromosome 7. Two genetic variants of the beta-subunit of the high-affinity IgE receptor (Fc epsilonRI-beta) gene were also analyzed. None of the asthmatic or control individuals carried the Ile181Leu variant. There were no significant differences between asthmatic and control subjects neither for the polymorphic markers nor for the other variant of the beta-subunit of the high-affinity IgE receptor (Fc epsilonRI-beta) gene. No association could be observed in this sample of Spanish asthmatics with the genes/regions studied.

  8. Isolation of cDNAs from the spinal muscular atrophy gene region with yeast artificial chromosomes

    SciTech Connect

    Deng, H.X.; He, X.X.; Hung, W.Y.

    1994-09-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of anterior horn cells, leading to progressive paralysis of voluntary muscles. The SMA gene(s) is located at 5q11.2-q13.3, between D5S435 and D5S112. To isolate potential candidate gene(s) responsible for SMA, we used the YACs within the SMA gene region as probes to screen a human brainstem cDNA library. Thirteen cDNA clones were isolated. Their sizes range from 0.7 kb to 5 kb. Seven clones were found to be unique in sequence; the remaining six clones contain repetitive sequences. Five out of these seven unique clones have been used as probes to screen a phage genomic DNA library. Phage genomic clones isolated with individual unique cDNA were used for fluorescence in situ hybridization to identify the origin of cDNAs. These five unique sequences are all located in the 5q13 region, indicating the reliability of our screening method. All the thirteen clones have been partially sequenced (about 300 bp) from each end. No homology has been found with any known EST or known genes. No cross hybridization was detected among the unique clones, suggesting that there may be distinct new genes encoded in this region.

  9. Sex chromosome loss and the pseudoautosomal region genes in hematological malignancies

    PubMed Central

    Weng, Stephanie; Stoner, Samuel A.; Zhang, Dong-Er

    2016-01-01

    Cytogenetic aberrations, such as chromosomal translocations, aneuploidy, and amplifications, are frequently detected in hematological malignancies. For many of the common autosomal aberrations, the mechanisms underlying their roles in cancer development have been well-characterized. On the contrary, although loss of a sex chromosome is observed in a broad range of hematological malignancies, how it cooperates in disease development is less understood. Nevertheless, it has been postulated that tumor suppressor genes reside on the sex chromosomes. Although the X and Y sex chromosomes are highly divergent, the pseudoautosomal regions are homologous between both chromosomes. Here, we review what is currently known about the pseudoautosomal region genes in the hematological system. Additionally, we discuss implications for haploinsufficiency of critical pseudoautosomal region sex chromosome genes, driven by sex chromosome loss, in promoting hematological malignancies. Because mechanistic studies on disease development rely heavily on murine models, we also discuss the challenges and caveats of existing models, and propose alternatives for examining the involvement of pseudoautosomal region genes and loss of a sex chromosome in vivo. With the widespread detection of loss of a sex chromosome in different hematological malignances, the elucidation of the role of pseudoautosomal region genes in the development and progression of these diseases would be invaluable to the field. PMID:27655702

  10. Pattern of diversity in the genomic region near the maize domestication gene tb1

    PubMed Central

    Clark, Richard M.; Linton, Eric; Messing, Joachim; Doebley, John F.

    2004-01-01

    Domesticated maize and its wild ancestor (teosinte) differ strikingly in morphology and afford an opportunity to examine the connection between strong selection and diversity in a major crop species. The tb1 gene largely controls the increase in apical dominance in maize relative to teosinte, and a region of the tb1 locus 5′ to the transcript sequence was a target of selection during maize domestication. To better characterize the impact of selection at a major “domestication” locus, we have sequenced the upstream tb1 genomic region and systematically sampled nucleotide diversity for sites located as far as 163 kb upstream to tb1. Our analyses define a selective sweep of ≈60–90 kb 5′ to the tb1 transcribed sequence. The selected region harbors a mixture of unique sequences and large repetitive elements, but it contains no predicted genes. Diversity at the nearest 5′ gene to tb1 is typical of that for neutral maize loci, indicating that selection at tb1 has had a minimal impact on the surrounding chromosomal region. Our data also show low intergenic linkage disequilibrium in the region and suggest that selection has had a minor role in shaping the pattern of linkage disequilibrium that is observed. Finally, our data raise the possibility that maize-like tb1 haplotypes are present in extant teosinte populations, and our findings also suggest a model of tb1 gene regulation that differs from traditional views of how plant gene expression is controlled. PMID:14701910

  11. Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions

    PubMed Central

    2010-01-01

    Background The lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment containing antibiotic cassette flanked by homology regions to the target locus into a strain that can express the lambda Red proteins (Gam, Bet, Exo). Results Here a scarless gene modification strategy based on the Red recombination system has been developed to modify Pseudomonas genome DNA via sequential deletion of multiple targets. This process was mediated by plasmid pRKaraRed encoding the Red proteins regulated by PBAD promoter, which was functional in P. aeruginosa as well as in other bacteria. First the target gene was substituted for the sacB-bla cassette flanked by short homology regions (50 bp), and then this marker gene cassette could be replaced by the PCR fragment flanking itself, generating target-deleted genome without any remnants and no change happened to the surrounding region. Twenty genes involved in the synthesis and regulation pathways of the phenazine derivate, pyocyanin, were modified, including one single-point mutation and deletion of two large operons. The recombination efficiencies ranged from 88% to 98%. Multiple-gene modification was also achieved, generating a triple-gene deletion strain PCA (PAO1, ΔphzHΔphzMΔphzS), which could produce another phenazine derivate, phenazine-1-carboxylic acid (PCA), efficiently and exclusively. Conclusions This lambda Red-based technique can be used to generate scarless and sequential gene modification mutants of P. aeruginosa efficiently, using one-step PCR product flanked by short homology regions. Single-point mutation, scarless deletion of genes can be achieved easily in less than three days. This method may give a new way to construct genetically modified P. aeruginosa strains more efficiently and advance the regulatory network study of this organism. PMID:20682065

  12. Nuclear reactor spacer grid and ductless core component

    DOEpatents

    Christiansen, David W.; Karnesky, Richard A.

    1989-01-01

    The invention relates to a nuclear reactor spacer grid member for use in a liquid cooled nuclear reactor and to a ductless core component employing a plurality of these spacer grid members. The spacer grid member is of the egg-shell type and is constructed so that the walls of the cell members of the grid member are formed of a single thickness of metal to avoid tolerance problems. Within each cell member is a hydraulic spring which laterally constrains the nuclear material bearing rod which passes through each cell member against a hardstop in response to coolant flow through the cell member. This hydraulic spring is also suitable for use in a water cooled nuclear reactor. A core component constructed of, among other components, a plurality of these spacer grid members, avoids the use of a full length duct by providing spacer sleeves about the sodium tubes passing through the spacer grid members at locations between the grid members, thereby maintaining a predetermined space between adjacent grid members.

  13. The evolution of spacers and valved holding chambers.

    PubMed

    Nikander, Kurt; Nicholls, Clare; Denyer, John; Pritchard, John

    2014-08-01

    Spacers and valved holding chambers (VHCs) are pressurized metered dose inhaler (pMDI) accessory devices, designed to overcome problems that patients commonly experience when administering aerosol via a pMDI. Spacers were developed in direct response to patient-related issues with pMDI technique, particularly, poor coordination between actuation and inhalation, and local side-effects arising from oropharyngeal deposition. Current clinical guidelines indicate the need for widespread prescription and use of spacers, but, despite their apparent ubiquity, the devices themselves are, unfortunately, all too commonly "disused" by patients. An understanding of the background from which spacers developed, and the key factors influencing the optimization of the spacer and the later VHC, is crucial to developing an appreciation of the potential of these devices, both contemporary and future, for improving the delivery of pressurized aerosols to patients. This review, informed by a full patent search and an extensive scientific literature review, takes into account the clinical and laboratory evidence, commercial developments, and the sometimes serendipitous details of scientific anecdotes to form a comprehensive perspective on the evolution of spacers, from their origins, in the early days of the pMDI, up to the present day.

  14. Sidewall spacer optimization for steep switching junctionless transistors

    NASA Astrophysics Data System (ADS)

    Gupta, Manish; Kranti, Abhinav

    2016-06-01

    In this work, we analyze the impact of a high permittivity (high-κ) sidewall spacer and gate dielectric on the occurrence of sub-60 mV/decade subthreshold swing (S-swing) in symmetrical junctionless (JL) double gate (DG) transistors. It is shown that steep S-swing values (≤10 mV/decade) can be achieved in JL devices with a combination of a high permittivity (high-κ) gate dielectric and a narrow low permittivity (low-κ) sidewall spacer. Implementation of a wider high-κ spacer will diminish the degree of impact ionization by the influence of the fringing component of the gate electric field, and will not be useful for steep off-to-on current transition. A wider spacer with low-κ and a narrow spacer with high-κ permittivity will be useful to limit the latching effect that can occur at lower temperatures (250 K). For high temperature operation, the decrease in the impact ionization rate can be compensated by designing a JL transistor with a thicker silicon film. The work demonstrates opportunities to enhance impact ionization at sub bandgap voltages, and proposes optimal guidelines for selecting a sidewall spacer to facilitate steep switching in JL transistors.

  15. Novel spacer device does not improve adherence in childhood asthma.

    PubMed

    Burgess, S W; Sly, P D; Cooper, D M; Devadason, S G

    2007-08-01

    The Funhaler (FH) is a novel spacer device (holding chamber) that has been designed to improve adherence and aerosol delivery in young asthmatic children using a metered dose inhaler. A pilot study reported a 38% increase in parent-reported adherence over 2 weeks compared with the child's normal spacer. The aim of this study was to investigate whether the FH would be associated with superior adherence in the medium term (3 months) using an objective assessment. Forty-seven children aged 18 months to 7 years were randomised to a FH or control small volume spacer. Participants were reviewed monthly for 3 months. Adherence was measured using an electronic monitoring device (Smartinhaler). Disease control was based on symptom scores and exacerbation rates. Twenty-six children were randomised to the FH and 21 to the control spacer. Three children withdrew (FH = 2). Median adherence each month for the 3 months was 74%, 54%, and 46% for the FH and 70%, 73%, and 54% for the control spacer. The difference in adherence was not statistically significant (P = 0.47, 0.37, and 0.23, respectively). There was also no significant difference in exacerbation rates or symptom scores. Seven of the FHs broke during the study. The FH was preferred by 21/24 parents randomised to the FH compared with their child's normal spacer. Despite the FH being popular with children and parents its use was not associated with improved adherence or disease control. 2007 Wiley-Liss, Inc.

  16. Analysis of tandem gene copies in maize chromosomal regions reconstructed from long sequence reads.

    PubMed

    Dong, Jiaqiang; Feng, Yaping; Kumar, Dibyendu; Zhang, Wei; Zhu, Tingting; Luo, Ming-Cheng; Messing, Joachim

    2016-07-19

    Haplotype variation not only involves SNPs but also insertions and deletions, in particular gene copy number variations. However, comparisons of individual genomes have been difficult because traditional sequencing methods give too short reads to unambiguously reconstruct chromosomal regions containing repetitive DNA sequences. An example of such a case is the protein gene family in maize that acts as a sink for reduced nitrogen in the seed. Previously, 41-48 gene copies of the alpha zein gene family that spread over six loci spanning between 30- and 500-kb chromosomal regions have been described in two Iowa Stiff Stalk (SS) inbreds. Analyses of those regions were possible because of overlapping BAC clones, generated by an expensive and labor-intensive approach. Here we used single-molecule real-time (Pacific Biosciences) shotgun sequencing to assemble the six chromosomal regions from the Non-Stiff Stalk maize inbred W22 from a single DNA sequence dataset. To validate the reconstructed regions, we developed an optical map (BioNano genome map; BioNano Genomics) of W22 and found agreement between the two datasets. Using the sequences of full-length cDNAs from W22, we found that the error rate of PacBio sequencing seemed to be less than 0.1% after autocorrection and assembly. Expressed genes, some with premature stop codons, are interspersed with nonexpressed genes, giving rise to genotype-specific expression differences. Alignment of these regions with those from the previous analyzed regions of SS lines exhibits in part dramatic differences between these two heterotic groups.

  17. Cytoplasmic polymorphism and evolutionary history of plum cultivars: Insights from chloroplast DNA sequence variation of trnL-trnF spacer and aggregated trnL intron & trnL-trnF spacer.

    PubMed

    Mustapha, S B; Ben Tamarzizt, H; Baraket, G; Abdallah, D; Salhi-Hannachi, A

    2015-04-27

    We screened for polymorphisms of the non-coding region of plastid DNA in plum trees. Sequencing data from the trnL-trnF chloroplast region were used to reveal a pattern of diversity, establish phylogenetic relationships, and test the selection pressure or evolutionary demography scenario for plastome DNA. The size of the non-coding regions varied from 398 to 563 and 865 to 1084 bases pairs for the trnL-trnF spacer and combined sequences, respectively. The average GC contents were 33.8 and 34.4% in the spacer and pooled sequences, respectively. Genetic distances calculated within the plums were 0.077 and 0.254, on average, for the trnL spacer and combined sequences, respectively. The neighbor-joining trees showed clustering relationships among cultivars that were independent of their geographic origins and designations. The neutrality tests and site-frequency spectra indicated that spacer and pooled sequences fit the neutral theory model at equilibrium between mutation and genetic drift and reject the hypothesis of a recent demographic expansion. The mismatch distribution shows variation patterns, thus providing evidence of an important genetic diversity explained by an excess of intermediate variants that occurred in the sequences analyzed. Further implications of the findings with regard to plum germplasm management and its utilization in breeding programs are also discussed.

  18. Methylation Analysis of the BMPR2 Gene Promoter Region in Patients With Pulmonary Arterial Hypertension.

    PubMed

    Pousada, Guillermo; Baloira, Adolfo; Valverde, Diana

    2016-06-01

    Pulmonary arterial hypertension is characterizated by obstruction of the pulmonary arteries. The gene mainly related to pathology is the bone morphogenetic protein receptor type II (BMPR2). The aim of this study was to analyze the methylation pattern of the BMPR2 promoter region in patients and controls. We used Methyl Primer Express(®) v.1.0 and MatInspector softwares to analyze this region. Genomic DNA obtained from the peripheral blood of patients and controls was modified with sodium bisulphite. Methylation was analyzed using methylation-specific PCR. DNA treated with CpG methyltransferase was used as a positive control for methylation and H1299 cell culture DNA was used as positive control for gene expression. We identified a CpG island, which may have been methylated, in the BMPR2 promoter region, in addition to NIT-2 (global-acting regulatory protein), sex-determining region Y) and heat shock factor transcription factor binding sites. We found no evidence of methylation in patients and controls. No methylated CpG sites were identified in H1299 cells expressing the BMPR2 gene. The BMPR2 promoter region is the most suitable for study because of the high number of transcription factor binding sites that could alter gene function. No evidence of methylation was detected in this region in patients and controls. Copyright © 2015 SEPAR. Published by Elsevier Espana. All rights reserved.

  19. RNA interference improves myopathic phenotypes in mice over-expressing FSHD region gene 1 (FRG1).

    PubMed

    Wallace, Lindsay M; Garwick-Coppens, Sara E; Tupler, Rossella; Harper, Scott Q

    2011-11-01

    Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders.

  20. Identification of Differentially Expressed Genes through Integrated Study of Alzheimer's Disease Affected Brain Regions.

    PubMed

    Puthiyedth, Nisha; Riveros, Carlos; Berretta, Regina; Moscato, Pablo

    2016-01-01

    Alzheimer's disease (AD) is the most common form of dementia in older adults that damages the brain and results in impaired memory, thinking and behaviour. The identification of differentially expressed genes and related pathways among affected brain regions can provide more information on the mechanisms of AD. In the past decade, several studies have reported many genes that are associated with AD. This wealth of information has become difficult to follow and interpret as most of the results are conflicting. In that case, it is worth doing an integrated study of multiple datasets that helps to increase the total number of samples and the statistical power in detecting biomarkers. In this study, we present an integrated analysis of five different brain region datasets and introduce new genes that warrant further investigation. The aim of our study is to apply a novel combinatorial optimisation based meta-analysis approach to identify differentially expressed genes that are associated to AD across brain regions. In this study, microarray gene expression data from 161 samples (74 non-demented controls, 87 AD) from the Entorhinal Cortex (EC), Hippocampus (HIP), Middle temporal gyrus (MTG), Posterior cingulate cortex (PC), Superior frontal gyrus (SFG) and visual cortex (VCX) brain regions were integrated and analysed using our method. The results are then compared to two popular meta-analysis methods, RankProd and GeneMeta, and to what can be obtained by analysing the individual datasets. We find genes related with AD that are consistent with existing studies, and new candidate genes not previously related with AD. Our study confirms the up-regualtion of INFAR2 and PTMA along with the down regulation of GPHN, RAB2A, PSMD14 and FGF. Novel genes PSMB2, WNK1, RPL15, SEMA4C, RWDD2A and LARGE are found to be differentially expressed across all brain regions. Further investigation on these genes may provide new insights into the development of AD. In addition, we identified

  1. Identification of Differentially Expressed Genes through Integrated Study of Alzheimer’s Disease Affected Brain Regions

    PubMed Central

    Berretta, Regina; Moscato, Pablo

    2016-01-01

    Background Alzheimer’s disease (AD) is the most common form of dementia in older adults that damages the brain and results in impaired memory, thinking and behaviour. The identification of differentially expressed genes and related pathways among affected brain regions can provide more information on the mechanisms of AD. In the past decade, several studies have reported many genes that are associated with AD. This wealth of information has become difficult to follow and interpret as most of the results are conflicting. In that case, it is worth doing an integrated study of multiple datasets that helps to increase the total number of samples and the statistical power in detecting biomarkers. In this study, we present an integrated analysis of five different brain region datasets and introduce new genes that warrant further investigation. Methods The aim of our study is to apply a novel combinatorial optimisation based meta-analysis approach to identify differentially expressed genes that are associated to AD across brain regions. In this study, microarray gene expression data from 161 samples (74 non-demented controls, 87 AD) from the Entorhinal Cortex (EC), Hippocampus (HIP), Middle temporal gyrus (MTG), Posterior cingulate cortex (PC), Superior frontal gyrus (SFG) and visual cortex (VCX) brain regions were integrated and analysed using our method. The results are then compared to two popular meta-analysis methods, RankProd and GeneMeta, and to what can be obtained by analysing the individual datasets. Results We find genes related with AD that are consistent with existing studies, and new candidate genes not previously related with AD. Our study confirms the up-regualtion of INFAR2 and PTMA along with the down regulation of GPHN, RAB2A, PSMD14 and FGF. Novel genes PSMB2, WNK1, RPL15, SEMA4C, RWDD2A and LARGE are found to be differentially expressed across all brain regions. Further investigation on these genes may provide new insights into the development of AD

  2. Analysis of ribosomal RNA intergenic spacer (IGS) sequences in species and populations of Gyrodactylus (Platyhelminthes: Monogenea) from salmonid fish in Northern Europe.

    PubMed

    Cunningham, C O; Collins, C M; Malmberg, G; Mo, T A

    2003-12-29

    The intergenic spacer (IGS) region of ribosomal RNA genes was amplified and sequenced from a variety of Gyrodactylus specimens collected from wild and farmed Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, and grayling Thymallus thymallus, from various locations in Northern Europe. Phylogenetic analysis of the sequences confirmed the distinction between G. salaris Malmberg, 1957 and G. thymalli Zitnan, 1960, supporting their validity as separate species. G. salaris adapted to rainbow trout are also distinct from the parasites found on Atlantic salmon, supporting the existence of a rainbow-trout form that was initially identified on the basis of morphological differences. Analysis of the IGS did not provide good resolution of different populations of G. salaris sensu stricto, but was consistent with epidemiological evidence which indicates that introduction of the parasite to Norway was recent and limited. The IGS may be helpful in distinguishing forms of G. salaris that are pathogenic to Atlantic salmon from those that are not.

  3. Characterization of a gene from the EDM1-PSACH region of human chromosome 19p

    SciTech Connect

    Lennon, G.G.; Giorgi, D.; Martin, J.R.

    1994-09-01

    Genetic linkage mapping has indicated that both multiple epiphyseal dysplasia (EDM1), a dominantly inherited chondrodysplasia, and pseudoachondroplasia (PSACH), a skeletal disorder associated with dwarfism, map to a 2-3 Mb region of human chromosome 19p. We have isolated a partial cDNA from this region using hybrid selection, and report on progress towards the characterization of the genomic structure and transcription of the corresponding gene. Sequence analysis of the cDNA to date indicates that this gene is likely to be expressed within extracellular matrix tissues. Defects in this gene or neighboring gene family members may therefore lead to EDM1, PSACH, or other connective tissue and skeletal disorders.

  4. PCR primers for 30 novel gene regions in the nuclear genomes of Lepidoptera

    PubMed Central

    Wahlberg, Niklas; Peña, Carlos; Ahola, Milla; Wheat, Christopher W.; Rota, Jadranka

    2016-01-01

    Abstract We report primer pairs for 30 new gene regions in the nuclear genomes of Lepidoptera that can be amplified using a standard PCR protocol. The new primers were tested across diverse Lepidoptera, including nonditrysians and a wide selection of ditrysians. These new gene regions give a total of 11,043 bp of DNA sequence data and they show similar variability to traditionally used nuclear gene regions in studies of Lepidoptera. We feel that a PCR-based approach still has its place in molecular systematic studies of Lepidoptera, particularly at the intrafamilial level, and our new set of primers now provides a route to generating phylogenomic datasets using traditional methods. PMID:27408580

  5. Information Theoretical Analysis of a Bovine Gene Atlas Reveals Chromosomal Regions with Tissue Specific Gene Expression.

    USDA-ARS?s Scientific Manuscript database

    An essential step to understanding the genomic biology of any organism is to comprehensively survey its transcriptome. We present the Bovine Gene Atlas (BGA) a compendium of over 7.2 million unique 20 base Illumina DGE tags representing 100 tissue transcriptomes collected primarily from L1 Dominette...

  6. Sequence Analysis of the Direct Repeat Region in Mycobacterium bovis

    PubMed Central

    Caimi, Karina; Romano, Maria I.; Alito, Alicia; Zumarraga, Martin; Bigi, Fabiana; Cataldi, Angel

    2001-01-01

    Spoligotyping is a major tool for molecular typing of Mycobacterium bovis. This technique is based on the polymorphism of spacers that separate direct repeats (DRs) in the M. tuberculosis complex DR region. Numerous M. bovis strains show a lack of several spacers which appears as a gap in the spoligotyping pattern. To determine whether these gaps contain alternative spacers not included in the spoligotyping membrane, PCRs using primers that hybridize to the spacers adjacent to the gaps were performed. Comparing the sizes of products obtained by PCR with those deduced from spoligotyping patterns, fragments were selected and sequenced to look for alternative spacers. Upon analysis of the sequences, five alternative spacers were detected, although deletions of spacers are mainly responsible for the observed gaps. The alternative spacers, which are more frequent in M. bovis than in M. tuberculosis, may contribute to increased M. bovis differentiation. PMID:11230428

  7. Diversity and Inheritance of Intergenic Spacer Sequences of 45S Ribosomal DNA among Accessions of Brassica oleracea L. var. capitata.

    PubMed

    Yang, Kiwoung; Robin, Arif Hasan Khan; Yi, Go-Eun; Lee, Jonghoon; Chung, Mi-Young; Yang, Tae-Jin; Nou, Ill-Sup

    2015-12-03

    Ribosomal DNA (rDNA) of plants is present in high copy number and shows variation between and within species in the length of the intergenic spacer (IGS). The 45S rDNA of flowering plants includes the 5.8S, 18S and 25S rDNA genes, the internal transcribed spacer (ITS1 and ITS2), and the intergenic spacer 45S-IGS (25S-18S). This study identified six different types of 45S-IGS, A to F, which at 363 bp, 1121 bp, 1717 bp, 1969 bp, 2036 bp and 2111 bp in length, respectively, were much shorter than the reported reference IGS sequences in B. oleracea var. alboglabra. The shortest two IGS types, A and B, lacked the transcription initiation site, non-transcribed spacer, and external transcribed spacer. Functional behavior of those two IGS types in relation to rRNA synthesis is a subject of further investigation. The other four IGSs had subtle variations in the transcription termination site, guanine-cytosine (GC) content, and number of tandem repeats, but the external transcribed spacers of these four IGSs were quite similar in length. The 45S IGSs were found to follow Mendelian inheritance in a population of 15 F₁s and their 30 inbred parental lines, which suggests that these sequences could be useful for development of new breeding tools. In addition, this study represents the first report of intra-specific (within subspecies) variation of the 45S IGS in B. oleracea.

  8. Diversity and Inheritance of Intergenic Spacer Sequences of 45S Ribosomal DNA among Accessions of Brassica oleracea L. var. capitata

    PubMed Central

    Yang, Kiwoung; Robin, Arif Hasan Khan; Yi, Go-Eun; Lee, Jonghoon; Chung, Mi-Young; Yang, Tae-Jin; Nou, Ill-Sup

    2015-01-01

    Ribosomal DNA (rDNA) of plants is present in high copy number and shows variation between and within species in the length of the intergenic spacer (IGS). The 45S rDNA of flowering plants includes the 5.8S, 18S and 25S rDNA genes, the internal transcribed spacer (ITS1 and ITS2), and the intergenic spacer 45S-IGS (25S-18S). This study identified six different types of 45S-IGS, A to F, which at 363 bp, 1121 bp, 1717 bp, 1969 bp, 2036 bp and 2111 bp in length, respectively, were much shorter than the reported reference IGS sequences in B. oleracea var. alboglabra. The shortest two IGS types, A and B, lacked the transcription initiation site, non-transcribed spacer, and external transcribed spacer. Functional behavior of those two IGS types in relation to rRNA synthesis is a subject of further investigation. The other four IGSs had subtle variations in the transcription termination site, guanine-cytosine (GC) content, and number of tandem repeats, but the external transcribed spacers of these four IGSs were quite similar in length. The 45S IGSs were found to follow Mendelian inheritance in a population of 15 F1s and their 30 inbred parental lines, which suggests that these sequences could be useful for development of new breeding tools. In addition, this study represents the first report of intra-specific (within subspecies) variation of the 45S IGS in B. oleracea. PMID:26633391

  9. The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe.

    PubMed

    Lorentz, A; Heim, L; Schmidt, H

    1992-06-01

    The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h90) strains of Schizosaccharomyces pombe. The MT region of h90 comprises three cassette genes: the expression site mat1:1 and two silent loci, mat2:2 and mat3:3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1:1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2:2 and mat3:3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.

  10. Characterization of the 5' flanking region of the human D1A dopamine receptor gene.

    PubMed Central

    Minowa, M T; Minowa, T; Monsma, F J; Sibley, D R; Mouradian, M M

    1992-01-01

    To study how the expression of the D1A dopamine receptor gene is regulated, a human genomic clone was isolated by using a rat cDNA as probe. A 2.3-kilobase genomic fragment spanning -2571 through -236 relative to the adenosine of the first methionine codon was sequenced. The gene has an intron of 116 base pairs in the 5' noncoding region, nucleotides -599 through -484 as determined by S1 mapping and reverse transcription-PCR. It has multiple transcription initiation sites located between -1061 and -1040. The promoter region lacks a TATA box and a CAAT box, is rich in G+C content, and has multiple putative binding sites for transcription factor Sp1. Thus, the promoter region of the human D1A gene has features of "housekeeping" genes. However, it also has consensus sequences for AP1 and AP2 binding sites and a putative cAMP response element. The ability of four deletion mutants of the 2.3-kilobase fragment to modulate transcription of the heterologous chloramphenicol acetyltransferase gene in the promoterless plasmid pCAT-Basic was determined. All mutants demonstrated substantial transcriptional activity in the murine neuroblastoma cell line NS20Y, which expresses the D1A gene endogenously. Transient expression assays suggested the presence of a positive modulator between nucleotides -1340 and -1102, and a negative modulator between -1730 and -1341. The four genomic fragments had no or very low transcriptional activity in NB41A3, C6, and Hep G2 cells, which are not known to express this gene. Thus, the human D1A gene belongs to the category of tissue-specific, regulated genes that have housekeeping-type promoters. Images PMID:1557411

  11. The genomic fingerprinting of the coding region of the beta-tubulin gene in Leishmania identification.

    PubMed

    Luis, L; Ramírez, A; Aguilar, C M; Eresh, S; Barker, D C; Mendoza-León, A

    1998-06-01

    We have demonstrated the polymorphism of the beta-tubulin gene region in Leishmania and its value in the identification of the parasite. In this work we have shown that the coding region of the gene has sufficient variation to accurately discriminate these parasites at the subgenus level. Nevertheless, intrasubgenus diversity, for particular restriction enzymes, was found in New World Leishmania belonging to the Leishmania subgenus. For instance, differences were found between mexicana and amazonensis strains. A unique pattern at the species level was found in particular species of both subgenera, e.g. L. (L.) major strain P and L. (L.) tropica belonging to the Leishmania subgenus, and L. (V.) panamensis strain LS94 from the Viannia subgenus. Particular endonucleases are diagnostic in Leishmania species discrimination as in the case of PvuII for the mexicana and amazonensis. This variation evidenced in the beta-tubulin gene region of Leishmania also occurred in other Kinetoplastida e.g. Trypanosoma cruzi, Leptomonas spp. and Crithidia spp. Moreover, these organisms showed a different genomic fingerprinting for the beta-tubulin gene among them and also Leishmania. Thus, the polymorphism of the coding region of the beta-tubulin gene can be used as a molecular marker for the identification of Leishmania.

  12. POLYMORPHISM IN THE CODING REGION SEQUENCE OF GDF8 GENE IN INDIAN SHEEP.

    PubMed

    Pothuraju, M; Mishra, S K; Kumar, S N; Mohamed, N F; Kataria, R S; Yadav, D K; Arora, R

    2015-11-01

    The present study was undertaken to identify polymorphism in the coding sequence of GDF8gene across indigenous meat type sheep breeds. A 1647 bp sequence was generated, encompassing 208 bp of the 5'UTR, 1128 bp of coding region (exon1, 2 and 3) as well as 311 bp of 3'UTR. The sheep and goat GDF8 gene sequences were observed to be highly conserved as compared to cattle, buffalo, horse and pig. Several nucleotide variations were observed across coding sequence of GDF8 gene in Indian sheep. Three polymorphic sites were identified in the 5'UTR, one in exon 1 and one in the exon 2 regions. Both SNPs in the exonic region were found to be non-synonymous. The mutations c.539T > G and c.821T > A discovered in this study in the exon 1 and exon 2, respectively, have not been previously reported. The information generated provides preliminary indication of the functional diversity present in Indian sheep at the coding region of GDF8gene. The novel as well as the previously reported SNPs discovered in the Indian sheep warrant further analysis to see whether they affect the phenotype. Future studies will need to establish the affect of reported SNPs in the expression of the GDF8 gene in Indian sheep population.

  13. The 5'-flanking regions of three pea legumin genes: comparison of the DNA sequences.

    PubMed Central

    Lycett, G W; Croy, R R; Shirsat, A H; Richards, D M; Boulter, D

    1985-01-01

    Approximately 1200 nucleotides of sequence data from the promoter and 5'-flanking regions of each of three pea (Pisum sativum L.) legumin genes (legA, legB and legC) are presented. The promoter regions of all three genes were found to be identical including the "TATA box", and "CAAT box', and sequences showing homology to the SV40 enhancers. The legA sequence begins to diverge from the others about 300bp from the start codon, whereas the other two genes remain identical for another 550bp. The regions of partial homology exhibit deletions or insertions and some short, comparatively well conserved sequences. The significance of these features is discussed in terms of evolutionary mechanisms and their possible functional roles. The legC gene contains a region that may potentially form either of two mutually exclusive stem-loop structures, one of which has a stem 42bp long, which suggests that it could be fairly stable. We suggest that a mechanism of switching between such alternative structures may play some role in gene control or may represent the insertion of a transposable element. PMID:2997721

  14. Core and region-enriched networks of behaviorally regulated genes and the singing genome

    PubMed Central

    Whitney, Osceola; Pfenning, Andreas R.; Howard, Jason T.; Blatti, Charles A; Liu, Fang; Ward, James M.; Wang, Rui; Audet, Jean-Nicolas; Kellis, Manolis; Mukherjee, Sayan; Sinha, Saurabh; Hartemink, Alexander J.; West, Anne E.; Jarvis, Erich D.

    2015-01-01

    Songbirds represent an important model organism for elucidating molecular mechanisms that link genes with complex behaviors, in part because they have discrete vocal learning circuits that have parallels with those that mediate human speech. We found that ~10% of the genes in the avian genome were regulated by singing, and we found a striking regional diversity of both basal and singing-induced programs in the four key song nuclei of the zebra finch, a vocal learning songbird. The region-enriched patterns were a result of distinct combinations of region-enriched transcription factors (TFs), their binding motifs, and presinging acetylation of histone 3 at lysine 27 (H3K27ac) enhancer activity in the regulatory regions of the associated genes. RNA interference manipulations validated the role of the calcium-response transcription factor (CaRF) in regulating genes preferentially expressed in specific song nuclei in response to singing. Thus, differential combinatorial binding of a small group of activity-regulated TFs and predefined epigenetic enhancer activity influences the anatomical diversity of behaviorally regulated gene networks. PMID:25504732

  15. Promoter region of the human platelet-derived growth factor A-chain gene.

    PubMed Central

    Takimoto, Y; Wang, Z Y; Kobler, K; Deuel, T F

    1991-01-01

    The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5' flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter region was exceptionally G + C-rich and contained a "TATA box" but no "CAAT box." The transcription start site was identified 845 base pairs 5' to the translation initiation site by S1 nuclease mapping and by primer extension. Both in vitro transcription and transient expression of the chloramphenicol acetyltransferase gene linked to the PDGF A-chain 5' flanking sequences established that the putative promoter region was active, and RNase H mapping established that the three characteristic mRNAs (1.9, 2.3, and 2.8 kilobases) used the same transcription start site, which was used in normal endothelial cells and in two human tumor cell lines that express high levels of A-chain transcripts. The results established an exceptionally G + C-rich promoter region and a single transcription start site active for each of the three mRNAs of the PDGF A-chain gene. DNA sites of potential importance in mediating the activation of the PDGF A-chain gene in normal cells and in transformed cell lines expressing high levels of PDGF A chain were identified. Images PMID:1848007

  16. Functional Interactions between Unlinked Muscle Genes within Haploinsufficient Regions of the Drosophila Genome

    PubMed Central

    Homyk-Jr., T.; Emerson-Jr., C. P.

    1988-01-01

    Mutations in 13 genes affecting muscle development in Drosophila have been examined in pairwise combinations for evidence of genetic interactions. Heterozygous combinations of mutations in five genes, including the gene coding for myosin heavy chain, result in more severe phenotypes than respective single heterozygous mutant controls. The various mutant interactions include examples showing allele-specific intergenic interactions, gene specific interactions, and allele-specific intragenic complementations, suggesting that some interactions result from the manner in which mutant gene products associate. Interactions that result from alterations in ``+'' gene copy number were also uncovered, suggesting that normal myofibril development requires that the relative amounts of respective gene products produced be tightly regulated. The importance of the latter parameter is substantiated by the finding that all five interacting loci map to disperse haploinsufficient or haplolethal regions of the genome. The implications of the present findings are discussed in relation to pursuing the phenomena involving genetic interactions to identify new genes encoding interacting myofibrillar proteins, to examine the nature of intermolecular interactions in mutant and normal development and to decipher the quantitative and temporal regulation of a large family of functionally related gene products. PMID:3135237

  17. Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

    PubMed

    Muñoz-Amatriaín, María; Lonardi, Stefano; Luo, MingCheng; Madishetty, Kavitha; Svensson, Jan T; Moscou, Matthew J; Wanamaker, Steve; Jiang, Tao; Kleinhofs, Andris; Muehlbauer, Gary J; Wise, Roger P; Stein, Nils; Ma, Yaqin; Rodriguez, Edmundo; Kudrna, Dave; Bhat, Prasanna R; Chao, Shiaoman; Condamine, Pascal; Heinen, Shane; Resnik, Josh; Wing, Rod; Witt, Heather N; Alpert, Matthew; Beccuti, Marco; Bozdag, Serdar; Cordero, Francesca; Mirebrahim, Hamid; Ounit, Rachid; Wu, Yonghui; You, Frank; Zheng, Jie; Simková, Hana; Dolezel, Jaroslav; Grimwood, Jane; Schmutz, Jeremy; Duma, Denisa; Altschmied, Lothar; Blake, Tom; Bregitzer, Phil; Cooper, Laurel; Dilbirligi, Muharrem; Falk, Anders; Feiz, Leila; Graner, Andreas; Gustafson, Perry; Hayes, Patrick M; Lemaux, Peggy; Mammadov, Jafar; Close, Timothy J

    2015-10-01

    Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

  18. T-cell receptor variable region gene usage in T-cell populations.

    PubMed Central

    Garman, R D; Ko, J L; Vulpe, C D; Raulet, D H

    1986-01-01

    We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens. Images PMID:3487085

  19. Effects of Elevated Source/Drain and Side Spacer Dielectric on the Drivability Optimization of Non-abrupt Ultra Shallow Junction Gate Underlap DG MOSFETs

    NASA Astrophysics Data System (ADS)

    Singh, Kunal; Kumar, Sanjay; Goel, Ekta; Singh, Balraj; Dubey, Sarvesh; Jit, Satyabrata

    2017-01-01

    The effects of drain/source elevation height ( h SD) and side spacer dielectric between the gate and source/drain regions on the drivability performance of the non-abrupt ultra-shallow-junction gate underlap double gate metal oxide field effect transistor of 18 nm gate length has been investigated in terms of the on-state ( I on), off-state ( I off) drain currents, and I on/ I off ratio. Among the Air, SiO2, Si3N4, and HfO2 used as spacer dielectrics, while both I on and I on/ I off are increased with the elevation height ( h SD) and permittivity of the spacer dielectric, interestingly, an inverse relation between the I on and I off for all h SD below ˜32.5 nm is observed only for the SiO2 spacer dielectric. Another new observation is the increase in I off with the h SD and permittivity of the spacer dielectric due to the enhancement of gate-induced drain leakage current owing to the increased vertical electric field at the drain side. For the most commonly used dielectrics SiO2 and HfO2 in the spacer region, the I on/ I off ratio is increased by ˜277% (at h SD = 32.5 nm) and ˜516% (at h SD = 9.5 nm) with respect to their corresponding values at zero elevation, respectively.

  20. Definition of a DNA repair domain in the genomic region containing the human p53 gene.

    PubMed

    Tolbert, D M; Kantor, G J

    1996-07-15

    The human p53 gene is repaired in UV (254 nm)-irradiated xeroderma pigmentosum group C (XP-C) cells as part of a large genomic region that is about twice the size of the gene. Surrounding genomic regions are not repaired. Through DNA cloning and measurements of DNA repair, we mapped the location of the repair domain, including the terminal regions, relative to the topological features of the gene. The domain includes only the DNA strand that is transcribed and extends in both 3' and 5' directions beyond the promoter and transcription termination sites. No transcriptional activity other than that associated with the p53 gene was detected. The results suggest that nontranscribed regions adjacent to the p53 transcribed regions are efficiently repaired in XP-C cells. This means that factors associated with transcription other than RNA polymerase II and the associated transcription repair coupling factor must also play a role in the selective repair process in XP-C cells. We also found that a DNA fragment that contains the p53 promoters is nearly twice as sensitive to cyclobutane pyrimidine dimer induction by UV irradiation than are the surrounding fragments, which have the expected sensitivity.

  1. The human insulin gene linked polymorphic region exhibits an altered DNA structure.

    PubMed Central

    Hammond-Kosack, M C; Dobrinski, B; Lurz, R; Docherty, K; Kilpatrick, M W

    1992-01-01

    Regulation of transcription of the human insulin gene appears to involve a series of DNA sequences in the 5' region. Hypersensitivity to DNA structural probes has previously been demonstrated in regulatory regions of cloned genomic DNA fragments, and been correlated with gene activity. To investigate the structure of the DNA in the human insulin gene, bromoacetaldehyde and S1 nuclease were reacted with a supercoiled plasmid containing a 5kb genomic insulin fragment. Both probes revealed the human insulin gene linked polymorphic region (ILPR), a region (-363) upstream of the transcriptional start site which contains multiple repeats of a 14-15mer oligonucleotide with the consensus sequence ACAGGGGT(G/C)(T/C)GGGG, as the major hypersensitive site. Fine mapping and electron microscopic analysis both show a very different behaviour of the two DNA strands in the region of the ILPR and suggest the G-rich strand may be adopting a highly structured conformation with the complementary strand remaining largely single stranded. Images PMID:1741248

  2. The identification of five novel genes in the cri-du-chat critical region

    SciTech Connect

    Simmons, A.D.; Gallardo, T.D.; Lovett, M.

    1994-09-01

    Cri-du-chat is a contiguous gene syndrome associated with deletions in the short arm of chromosome 5 (chr 5). Chr 5p-specific markers have been used to define two critical regions: a larynx malformation region, located at 5p15.3, and a region responsible for the remaining clinical features of the syndrome, which maps to 5p15.2. Thirty cosmids that map to this latter region have been isolated from the LANL chr 5-specific library using 5 STSs. More recently, we have constructed a YAC contig of the region which encompasses 2-3 Mb. The 30 framework cosmids were used in a direct selection with cDNAs from placenta, activated T-cells and cerebellum to isolate an initial set of expressed sequences from this region. Since no genes, to date, have been isolated or localized within the cri-du-chat deletion, a cosmid containing a control reporter gene (ANX6) was used to monitor enrichment. ANX6 cDNAs were enriched by several thousand-fold in the selected cDNAs. A total of nine non overlapping cDNA fragments were obtained from the cDNA pools. These have been ordered within the YAC contig, map to 5 discrete cosmid sets in the critical region and thus conservatively represent five discrete transcription units. The DNA sequences of these fragments are novel by sequence database comparisons. PCR primers were constructed and were used to confirm gene placements in the YAC contig, as well as to investigate the expression profile of these genes in several different tissues and cell types. In one case, these primer sets enabled two of the nine fragments to be linked into a larger cDNA. The nine cDNAs showed various patterns of differential expression in a panel of tissues. These expressed sequences represent the first genes isolated within the cri-du-chat critical region and represent the initial steps in the derivation of a comprehensive inventory and expression profile of the estimated 100 genes that may reside in this region.

  3. Analysis of spatial-temporal gene expression patterns reveals dynamics and regionalization in developing mouse brain.

    PubMed

    Chou, Shen-Ju; Wang, Chindi; Sintupisut, Nardnisa; Niou, Zhen-Xian; Lin, Chih-Hsu; Li, Ker-Chau; Yeang, Chen-Hsiang

    2016-01-20

    Allen Brain Atlas (ABA) provides a valuable resource of spatial/temporal gene expressions in mammalian brains. Despite rich information extracted from this database, current analyses suffer from several limitations. First, most studies are either gene-centric or region-centric, thus are inadequate to capture the superposition of multiple spatial-temporal patterns. Second, standard tools of expression analysis such as matrix factorization can capture those patterns but do not explicitly incorporate spatial dependency. To overcome those limitations, we proposed a computational method to detect recurrent patterns in the spatial-temporal gene expression data of developing mouse brains. We demonstrated that regional distinction in brain development could be revealed by localized gene expression patterns. The patterns expressed in the forebrain, medullary and pontomedullary, and basal ganglia are enriched with genes involved in forebrain development, locomotory behavior, and dopamine metabolism respectively. In addition, the timing of global gene expression patterns reflects the general trends of molecular events in mouse brain development. Furthermore, we validated functional implications of the inferred patterns by showing genes sharing similar spatial-temporal expression patterns with Lhx2 exhibited differential expression in the embryonic forebrains of Lhx2 mutant mice. These analysis outcomes confirm the utility of recurrent expression patterns in studying brain development.

  4. Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells.

    PubMed

    Takami, Nozomi; Osawa, Kayo; Miura, Yasushi; Komai, Koichiro; Taniguchi, Mariko; Shiraishi, Masahiko; Sato, Keizo; Iguchi, Tetsuhiro; Shiozawa, Kazuko; Hashiramoto, Akira; Shiozawa, Shunichi

    2006-03-01

    To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.

  5. New genes in the class II region of the human major histocompatibility complex.

    PubMed

    Hanson, I M; Poustka, A; Trowsdale, J

    1991-06-01

    A detailed map of the class II region of the human major histocompatibility complex has been constructed by pulsed-field gel electrophoresis. This map revealed clusters of sites for enzymes that cut preferentially in unmethylated CpG-rich DNA often found at the 5' ends of genes. Three of these clusters have been cloned by cosmid walking and chromosome jumping. Analysis of the clones encompassing these regions through the use of zoo blots, Northern blots, and cDNA libraries resulted in the discovery of four novel genes. The D6S111E and D6S112E genes are centromeric to the HLA-DPB2 gene, while D6S113E and D6S114E are between HLA-DNA and HLA-DOB. Preliminary characterization of the new genes indicates that they are unrelated to the class II genes themselves, although D6S114E expression, like class II expression, is inducible with interferon. In addition, the HLA-DNA gene has been accurately positioned and oriented for the first time.

  6. Polymorphism in the upstream regulatory region of DQA1 gene in the Italian population.

    PubMed

    Petronzelli, F; Kimura, A; Ferrante, P; Mazzilli, M C

    1995-04-01

    Polymorphism in the 5'-upstream regulatory region of the DQA1 gene has been recently described. Using PCR-SSO method and SSCP analysis we have investigated this polymorphism in a group of 111 Italian blood donors which had been oligotyped for DRB1, DQA1 and DQB1 genes. Eight allelic variants were detected. Looking at the relationships among QAP sequences and DQA1 and DRB1 genes, three alternative situations were found: 1. a one-to-one relation between QAP and DQA1 alleles, independently of the other class II genes; 2. the same QAP allele in association with different DQA1-DRB1 haplotypes; 3. the same DQA1 allele with different QAP sequences according to the DRB1 specificity. No unexpected associations with DQB1 gene were found. These results must be interpreted considering that DQA1 and DRB1 genes are transcribed in opposite directions so that the promoter region of DQA1 gene lies between DQA1 and DRB1, close to the former but several hundreds kb away from the latter.

  7. Perforation of the sigmoid colon due to intradiscal spacer dislocation.

    PubMed

    Ruf, Michael; Voigt, Andreas; Kupczyk-Joeris, Dieter; Merk, Harry R

    2011-07-01

    A case of late dislocation of a disc spacer L5/S1 with perforation of the sigmoid colon and transanal passage 4 years after implantation is reported. The objective is to describe an uncommon complication of anterior endoscopic spondylodesis L5/S1. To our knowledge, this is the first report on this rare complication. A 39-year-old patient suffering from a spondylolisthesis L5/S1 (Meyerding grade 2) with bilateral lysis L5 was operated with posterior instrumentation L5/S1 and anterior endoscopic insertion of two disc spacers. 4 years after surgery the patient noticed one of the spacers in the toilet. Radiographic examination of the colon with contrast dye revealed a perforation at the distal sigmoid colon. At the lumbosacral junction there was a bony defect at the site of the absent spacer and an anterior dislocation of the second spacer. A partial resection of the colon at the perforation site with end-to-end anastomosis was performed. The second spacer was removed, and the defect was packed with autologous cancellous bone and local antibiotics. The further course was uneventful. 2 weeks postoperatively the patient was discharged without signs of infection. The radiographic examination after 6 months showed healing of the bone graft with bony fusion L5/S1. In case of incomplete or absent bony fusion the dislocation of intradiscal spacers may arise even years after the primary surgery. In consequence periodical radiographic examinations of spinal instrumentations are recommended until complete bony fusion occurred. Unclear abdominal symptoms following anterior spine surgery require immediate examination.

  8. Reducing electrostatic charge on spacer devices and bronchodilator response

    PubMed Central

    Wildhaber, Johannes H; Waterer, Grant W; Hall, Graham L; Summers, Quentin A

    2000-01-01

    Aims Plastic spacers are widely used with pressurized metered dose inhalers (pMDI). Reducing electrostatic charge by washing spacers with detergent has been shown to greatly improve in vitro and in vivo drug delivery. We assessed whether this finding is associated with an improved bronchodilator response in adult asthmatics. Methods Twenty subjects (age 18–65 years) with a known bronchodilator response inhaled in random order salbutamol from a pMDI (Ventolin®) through an untreated new spacer (Volumatic®) and through a detergent washed spacer. Patients received the following doses of salbutamol via pMDI at 20 min intervals: 100 µg, 100 µg, 200 µg, 400 µg, 800 µg. Spirometry, heart rate and blood pressure were checked prior to each dose and 20 min after the last dose. Results There were no differences between baseline forced expiratory volume in 1 s (FEV1) using either spacer (2.61 ± 0.56 and 2.52 ± 0.45 l, untreated and treated with detergent, respectively; mean ±s.d.). The provocation dose required to cause a clinically significant improvement of 10% in FEV1 (PD10) was significantly lower when the detergent treated spacer was used (1505 ± 1335 and 430 ± 732 µg, untreated and treated, respectively, P < 0.002). Conclusions We have demonstrated an improvement in bronchodilator response, in adult asthmatics, after reducing the electrostatic charge in a spacer device by washing it with ordinary household detergent. This finding stresses the importance of an optimal choice of delivery device for asthma medication. PMID:10971314

  9. Histone deacetylase inhibition modulates histone acetylation at gene promoter regions and affects genome-wide gene transcription in Schistosoma mansoni

    PubMed Central

    Anderson, Letícia; Gomes, Monete Rajão; daSilva, Lucas Ferreira; Pereira, Adriana da Silva Andrade; Mourão, Marina M.; Romier, Christophe; Pierce, Raymond

    2017-01-01

    Background Schistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known. Methodology/Principal findings TSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality. Conclusions/Significance Genome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression

  10. Histone deacetylase inhibition modulates histone acetylation at gene promoter regions and affects genome-wide gene transcription in Schistosoma mansoni.

    PubMed

    Anderson, Letícia; Gomes, Monete Rajão; daSilva, Lucas Ferreira; Pereira, Adriana da Silva Andrade; Mourão, Marina M; Romier, Christophe; Pierce, Raymond; Verjovski-Almeida, Sergio

    2017-04-01

    Schistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known. TSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality. Genome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression of dozens of histone reader genes involved in regulation of the

  11. Role of spacer lengths of gemini surfactants in the synthesis of silver nanorods in micellar media.

    PubMed

    Bhattacharya, Santanu; Biswas, Joydeep

    2011-07-01

    In this work, we have prepared Ag-nanorods using biscationic gemini surfactant micelles as the media by a seed-mediated wet synthesis method. Towards this end, we first synthesized Ag-nanoseeds of diameter ~7 nm stabilized by trisodium citrate (as the capping agent). Then these Ag-nanoseeds were used to synthesize Ag-nanorods of different aspect ratios. With decreasing Ag-nanoseed concentration, the aspect ratios of the Ag-nanorods stabilized by these gemini surfactants increased gradually. Various Ag-nanoseeds and Ag-nanospecies were characterized using UV-Vis spectroscopy (to know the surface plasmon bands), transmission electron microscopy (to find out their particle sizes and distribution), energy-dispersive X-ray spectroscopy and X-ray diffraction. When we used micelles derived from gemini surfactants of shorter spacer -(CH(2))(n)- (n = 2 or 4) to stabilize the Ag-nanorods, the λ(max) of the longitudinal band shifted more towards the blue region compared to that of the gemini surfactant micelles with a longer spacer -(CH(2))(n)- (n = 5, 12) at a given amount of the Ag-nanoseed solution. So, the growth of Ag-nanorods in the gemini micellar solutions depends on the spacer-chain length of gemini surfactants employed.

  12. Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity

    PubMed Central

    Nuñez, James K.; Lee, Amy S.Y.; Engelman, Alan; Doudna, Jennifer A.

    2015-01-01

    Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit, whereas Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting A-T rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyzes spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity. PMID:25707795

  13. Human heavy-chain variable region gene family nonrandomly rearranged in familial chronic lymphocytic leukemia

    SciTech Connect

    Shen, A.; Humphries, C.; Tucker, P.; Blattner, F.

    1987-12-01

    The authors have identified a family of human immunoglobulin heavy-chain variable-region (V/sub H/) genes, one member of which is rearranged in two affected members of a family in which the father and four of five siblings developed chronic lymphocytic leukemia. Cloning and sequencing of the rearranged V/sub H/ genes from leukemic lymphocytes of three affected siblings showed that two siblings had rearranged V/sub H/ genes (V/sub H/TS1 and V/sub H/WS1) that were 90% homologous. The corresponding germ-line gene, V/sub H/251, was found to part of a small (four gene) V/sub H/ gene family, which they term V/sub H/V. The DNA sequence homology to V/sub H/WS1 (95%) and V/sub H/TS1 (88%) and identical restriction sites on the 5' side of V/sub H/ confirm that rearrangement of V/sub H/251 followed by somatic mutation produced the identical V/sub H/ gene rearrangements in the two siblings. V/sub H/TS1 is not a functional V/sub H/ gene; a functional V/sub H/ rearrangement was found on the other chromosome of this patient. The other two siblings had different V/sub H/ gene rearrangements. All used different diversity genes. Mechanisms proposed for nonrandom selection of a single V/sub H/ gene include developmental regulation of this V/sub H/ gene rearrangement or selection of a subpopulation of B cells in which this V/sub H/ has been rearranged.

  14. Differential gene expression in mouse retina related to regional differences in vulnerability to hyperoxia

    PubMed Central

    Natoli, Riccardo; Valter, Krisztina; Stone, Jonathan

    2010-01-01

    Purpose In the C57BL/6J mouse retina, hyperoxia-induced degeneration of photoreceptors shows strong regional variation, beginning at a locus ~0.5 mm inferior to the optic disc. To identify gene expression differences that might underlie this variability in vulnerability, we have used microarray techniques to describe regional (superior-inferior) variations in gene expression in the retina. Methods Young adult C57BL/6J mice raised in dim cyclic illumination (12 h at 5 lx and 12 h in darkness) were exposed to hyperoxia (75% oxygen for two weeks). Retinas were collected from hyperoxia-exposed and control animals without fixation and divided into superior and inferior halves. RNA was extracted from each sample, purified, and hybridized to Mouse Gene 1.0 ST arrays (Affymetrix). The consistency of the microarray results was assessed using quantitative PCR for selected genes. Expression data were analyzed to identify genes and ncRNAs whose differential expression between the superior and inferior retina could be associated with relative vulnerability to hyperoxia. Results In control retinas, only two genes showed a fold difference in expression >2 between the superior and inferior retina; another 25 showed a fold difference of 1.5–2.0. Of these 27, the functions of six genes, including ventral anterior homeobox containing gene 2 (Vax2) and T-box 5 (Tbox5), are related to parameters of anatomic development and the functions of five are related to sensory perception. Among the latter, short-wave-sensitive cone opsin (Opn1sw) was more strongly expressed in the inferior retina and medium-wave-sensitive cone opsin (Opn1mw) in the superior retina. This is consistent with known differences in S- and M-cone distribution, confirming our separation of retinal regions. The highest fold difference was reported for membrane metalloendopeptidase (Mme), a member from the metallothionein group of cytoprotective proteins. To identify genes whose regulation by hyperoxia was

  15. Utilizing ribosomal DNA gene marker regions to characterize the metacercariae (Trematoda: Digenea) parasitizing piscine intermediate hosts in Manipur, Northeast India.

    PubMed

    Athokpam, Voleentina D; Jyrwa, Donald B; Tandon, Veena

    2016-06-01

    Freshwater fishes in Manipur, Northeast India frequently harbour several types of metacercariae, which based on morphological criteria were identified as Clinostomoides brieni, Euclinostomum heterostomum (Clinostomidae) and Polylekithum sp. (Allocreadiidae). Molecular techniques utilizing PCR amplification of rDNA regions of larger subunit (LSU or 28S), smaller subunit (SSU or 18S) and inter transcribed spacers (ITS1, 2) were used for molecular characterization of these types. Sequences generated from the metacercariae were compared with their related sequences available in public databases; an analysis of the identity matrices and phylogenetic trees constructed was also carried out, which confirmed their identification. Similarly, the sequences generated from Polylekithum sp. were found to be highly similar to the species of the same genus. The rDNA ITS2 secondary structure provided additional confirmation of the robustness of the molecular marker as a tool for taxon-specific characterization.

  16. Sensitivity and correlation of hypervariable regions in 16S rRNA genes in phylogenetic analysis.

    PubMed

    Yang, Bo; Wang, Yong; Qian, Pei-Yuan

    2016-03-22

    Prokaryotic 16S ribosomal RNA (rRNA) sequences are widely used in environmental microbiology and molecular evolution as reliable markers for the taxonomic classification and phylogenetic analysis of microbes. Restricted by current sequencing techniques, the massive sequencing of 16S rRNA gene amplicons encompassing the full length of genes is not yet feasible. Thus, the selection of the most efficient hypervariable regions for phylogenetic analysis and taxonomic classification is still debated. In the present study, several bioinformatics tools were integrated to build an in silico pipeline to evaluate the phylogenetic sensitivity of the hypervariable regions compared with the corresponding full-length sequences. The correlation of seven sub-regions was inferred from the geodesic distance, a parameter that is applied to quantitatively compare the topology of different phylogenetic trees constructed using the sequences from different sub-regions. The relationship between different sub-regions based on the geodesic distance indicated that V4-V6 were the most reliable regions for representing the full-length 16S rRNA sequences in the phylogenetic analysis of most bacterial phyla, while V2 and V8 were the least reliable regions. Our results suggest that V4-V6 might be optimal sub-regions for the design of universal primers with superior phylogenetic resolution for bacterial phyla. A potential relationship between function and the evolution of 16S rRNA is also discussed.

  17. Functional characterization of genetic polymorphisms identified in the promoter region of the bovine PEPS gene.

    PubMed

    Ju, Zhihua; Zheng, Xue; Huang, Jinming; Qi, Chao; Zhang, Yan; Li, Jianbin; Zhong, Jifeng; Wang, Changfa

    2012-06-01

    Peptidase S (PEPS) is a metallopeptidase that cleaves N-terminal residues from proteins and peptides. PEPS is used as a cell maintenance enzyme with critical roles in peptide turnover. The promoter region located upstream of the initiation site plays an important role in regulating gene expression. Polymorphism in the promoter region can alter gene expression and lead to biological changes. In the current study, polymorphisms in the promoter region of the PEPS gene were investigated. Polymerase chain reaction (PCR)-restriction fragment length polymorphism and DNA sequencing methods were used to screen sequence variations in the promoter region of DNA samples from 743 Chinese Holstein cattle. Two polymorphisms (g. -534 T>C and g. -2545 G>A) were identified and eight haplotypes were classified by haplotype analysis. The two genetic polymorphisms and haplotypes were associated with fat percentage and somatic cell score in Chinese Holstein cattle. The results of real-time PCR showed that cow kidneys exhibit the highest PEPS expression level. Moreover, bioinformatics analysis predicted that the single-nucleotide polymorphism g. -534 T>C is located in the core promoter region and in the transcription factor binding sites. The promoter activities of the polymorphism of -543 T>C were measured by luciferase assay in the human kidney epithelial cell line 293T. Transcriptional activity is significantly lower in cell lines transfected with the reporter construct containing 2.5 kb upstream fragments with -543 C than in those with wild-type -543 T. The results indicated that genetic variation at locus -543 influences PEPS promoter activity. The genetic variation in the promoter region of PEPS gene may regulate PEPS gene transcription and might have consequences at a regulatory level.

  18. Role of Helicobacter pylori plasticity region genes in development of gastroduodenal diseases.

    PubMed

    Sugimoto, Mitsushige; Watada, Masahide; Jung, Sung Woo; Graham, David Y; Yamaoka, Yoshio

    2012-02-01

    The plasticity region of Helicobacter pylori is a large chromosomal segment including isolate-specific open reading frames with characteristics of pathogenicity islands. It remains unclear whether genes in the plasticity region play a role in the pathogenesis of gastric mucosal inflammation and gastroduodenal disease. Our aim was to assess the role of selected genes in the plasticity region in relation to risk of H. pylori-related disease and the severity of gastric mucosal damage. We used PCR to study the relation of disease outcome and mucosal damage with four genes in the H. pylori plasticity region (jhp0940, jhp0945, jhp0947, and jhp0949) from isolates obtained from both Western (n = 296) and East Asian (n = 217) patients. The prevalence of jhp0945, jhp0947, and jhp0949 differed significantly between Western and East Asian isolates. In Western isolates, the presence of jhp0945 was significantly associated with gastric ulcer, duodenal ulcer, and gastric cancer (odds ratios [95% confidence intervals]: 2.27 [1.04 to 4.98], 1.86 [1.03 to 3.34], and 1.92 [1.03 to 3.56], respectively). jhp0940-positive Western isolates were significantly associated with absence of gastric ulcer or duodenal ulcer (0.21 [0.05 to 0.94] and 0.31 [0.12 to 0.78], respectively). No significant difference was observed between inflammatory cell infiltration or atrophy and the presence or absence of plasticity region genes. The outcome of H. pylori infections varies widely geographically. These data suggest a possible role for difference in the prevalence of plasticity region genes in the geographic variation in H. pylori-related diseases.

  19. Arrangement and number of clustered regularly interspaced short palindromic repeat spacers are associated with erythromycin susceptibility in emm12, emm75 and emm92 of group A streptococcus.

    PubMed

    Zheng, P-X; Chiang-Ni, C; Wang, S-Y; Tsai, P-J; Kuo, C-F; Chuang, W-J; Lin, Y-S; Liu, C-C; Wu, J-J

    2014-06-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) are composed of numerous repeat-spacer units and are considered a prokaryotic defence system against foreign nucleic acids. Since antibiotic-resistant genes are frequently encoded in foreign nucleic acids, the aim of this study was to test whether erythromycin susceptibility in group A streptococcus (Streptococcus pyogenes) is associated with characteristics of CRISPR elements. Erythromycin susceptibility of 330 isolates collected between 1997 and 2003 was analysed. Among 29 emm types, emm12, emm75 and emm92 showed significant changes in erythromycin-resistance rates. By sequencing the spacers from two CRISPR loci, spacer contents in emm12, emm75 and emm92 strains were associated with erythromycin susceptibility. Strains with fewer spacers were more resistant to erythromycin. Moreover, in emm4 strains, which showed no significant change in their annual erythromycin-resistance rate, CRISPR type and number of spacers were not correlated with erythromycin susceptibility. These results highlight a novel association between CRISPR spacer content and erythromycin susceptibility in group A streptococcus. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  20. Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

    PubMed Central

    Daley, M D; Misener, V; Olee, T; Chen, P P; Siminovitch, K A

    1993-01-01

    Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection. PMID:8324896

  1. Global differential expression of genes located in the Down Syndrome Critical Region in normal human brain

    PubMed Central

    Montoya, Julio Cesar; Fajardo, Dianora; Peña, Angela; Sánchez, Adalberto; Domínguez, Martha C; Satizábal, José María

    2014-01-01

    Background: The information of gene expression obtained from databases, have made possible the extraction and analysis of data related with several molecular processes involving not only in brain homeostasis but its disruption in some neuropathologies; principally in Down syndrome and the Alzheimer disease. Objective: To correlate the levels of transcription of 19 genes located in the Down Syndrome Critical Region (DSCR) with their expression in several substructures of normal human brain. Methods: There were obtained expression profiles of 19 DSCR genes in 42 brain substructures, from gene expression values available at the database of the human brain of the Brain Atlas of the Allen Institute for Brain Sciences", (http://human.brain-map.org/). The co-expression patterns of DSCR genes in brain were calculated by using multivariate statistical methods. Results: Highest levels of gene expression were registered at caudate nucleus, nucleus accumbens and putamen among central areas of cerebral cortex. Increased expression levels of RCAN1 that encode by a protein involved in signal transduction process of the CNS were recorded for PCP4 that participates in the binding to calmodulin and TTC3; a protein that is associated with differentiation of neurons. That previously identified brain structures play a crucial role in the learning process, in different class of memory and in motor skills. Conclusion: The precise regulation of DSCR gene expression is crucial to maintain the brain homeostasis, especially in those areas with high levels of gene expression associated with a remarkable process of learning and cognition. PMID:25767303

  2. Global differential expression of genes located in the Down Syndrome Critical Region in normal human brain.

    PubMed

    Montoya, Julio Cesar; Fajardo, Dianora; Peña, Angela; Sánchez, Adalberto; Domínguez, Martha C; Satizábal, José María; García-Vallejo, Felipe

    2014-01-01

    The information of gene expression obtained from databases, have made possible the extraction and analysis of data related with several molecular processes involving not only in brain homeostasis but its disruption in some neuropathologies; principally in Down syndrome and the Alzheimer disease. To correlate the levels of transcription of 19 genes located in the Down Syndrome Critical Region (DSCR) with their expression in several substructures of normal human brain. There were obtained expression profiles of 19 DSCR genes in 42 brain substructures, from gene expression values available at the database of the human brain of the Brain Atlas of the Allen Institute for Brain Sciences", (http://human.brain-map.org/). The co-expression patterns of DSCR genes in brain were calculated by using multivariate statistical methods. Highest levels of gene expression were registered at caudate nucleus, nucleus accumbens and putamen among central areas of cerebral cortex. Increased expression levels of RCAN1 that encode by a protein involved in signal transduction process of the CNS were recorded for PCP4 that participates in the binding to calmodulin and TTC3; a protein that is associated with differentiation of neurons. That previously identified brain structures play a crucial role in the learning process, in different class of memory and in motor skills. The precise regulation of DSCR gene expression is crucial to maintain the brain homeostasis, especially in those areas with high levels of gene expression associated with a remarkable process of learning and cognition.

  3. Stochastic rearrangement of immunoglobulin variable-region genes in chicken B-cell development.

    PubMed

    Benatar, T; Tkalec, L; Ratcliffe, M J

    1992-08-15

    The molecular mechanism by which immunoglobulin (Ig) gene rearrangement occurs is highly conserved between mammalian and avian species. However, in avian species, an equivalent to the mammalian pre-B cell, which has undergone Ig heavy-chain gene rearrangement and expresses mu heavy chains in the absence of Ig light-chain rearrangement, has not been convincingly demonstrated. It is consequently unclear whether an ordered progression of gene rearrangement events leading to functional Ig expression occurs in avian species. To examine the sequence of Ig gene rearrangement events in chicken B-cell development, we transformed day 12 embryo bursal cells with the REV-T(CSV) retrovirus. More than 100 clones were analyzed by Southern blotting and polymerase chain reaction for the presence of Ig gene rearrangements. The majority of these clones contained only germline Ig sequences. Several clones contained complete heavy- and light-chain rearrangements and 13 clones contained only heavy-chain rearrangements analogous to stages of mammalian B-cell development. However, 5 clones contained rearrangements of light-chain genes in the absence of complete heavy-chain rearrangement. Consequently, we conclude that rearrangement of chicken Ig light-chain genes does not require heavy-chain variable-region rearrangement. This observation suggests that chicken Ig gene rearrangement events required for Ig expression occur stochastically rather than sequentially.

  4. Voltage-time characteristics for steep-front impulse voltages of particle-contaminated spacers in SF sub 6 gas-insulated switchgear

    SciTech Connect

    Okabe, S.; Yorozuya, T. ); Endo, F.; Yamagiwa, T.; Iwassa, S. )

    1992-07-01

    In this paper voltage-time characteristics of spacer surfaces for steep-front impulse waves are investigated under a particle-contaminated condition in SF{sub 6} gas. The characteristics are measured as a function of particle length, particle position and spacer shapes. Flashover voltages monotonically increase in the submicrosecond region as time to flashover is shorter, and are a minimum in the 1 {mu}s region. Applicability of the equal voltage-time area criterion for estimating voltage-time characteristics is discussed and the technical meaning of the two parameters used in the estimation is clarified. Moreover, it is demonstrated that an optimized spacer with ribs remarkably improves flashover voltages in the submicrosecond region as well as in the 1 {mu}s and power frequency region.

  5. Conservation of sequence in recombination signal sequence spacers.

    PubMed Central

    Ramsden, D A; Baetz, K; Wu, G E

    1994-01-01

    The variable domains of immunoglobulins and T cell receptors are assembled through the somatic, site specific recombination of multiple germline segments (V, D, and J segments) or V(D)J rearrangement. The recombination signal sequence (RSS) is necessary and sufficient for cell type specific targeting of the V(D)J rearrangement machinery to these germline segments. Previously, the RSS has been described as possessing both a conserved heptamer and a conserved nonamer motif. The heptamer and nonamer motifs are separated by a 'spacer' that was not thought to possess significant sequence conservation, however the length of the spacer could be either 12 +/- 1 bp or 23 +/- 1 bp long. In this report we have assembled and analyzed an extensive data base of published RSS. We have derived, through extensive consensus comparison, a more detailed description of the RSS than has previously been reported. Our analysis indicates that RSS spacers possess significant conservation of sequence, and that the conserved sequence in 12 bp spacers is similar to the conserved sequence in the first half of 23 bp spacers. PMID:8208601

  6. Impact of spacer thickness on biofouling in forward osmosis.

    PubMed

    Valladares Linares, R; Bucs, Sz S; Li, Z; AbuGhdeeb, M; Amy, G; Vrouwenvelder, J S

    2014-06-15

    Forward osmosis (FO) indirect desalination systems integrate wastewater recovery with seawater desalination. Niche applications for FO systems have been reported recently, due to the demonstrated advantages compared to conventional high-pressure membrane processes such as nanofiltration (NF) and reverse osmosis (RO). Among them, wastewater recovery has been identified to be particularly suitable for practical applications. However, biofouling in FO membranes has rarely been studied in applications involving wastewater effluents. Feed spacers separating the membrane sheets in cross-flow systems play an important role in biofilm formation. The objective of this study was to determine the influence of feed spacer thickness (28, 31 and 46 mil) on biofouling development and membrane performance in a FO system, using identical cross-flow cells in parallel studies. Flux development, biomass accumulation, fouling localization and composition were determined and analyzed. For all spacer thicknesses, operated at the same feed flow and the same run time, the same amount of biomass was found, while the flux reduction decreased with thicker spacers. These observations are in good agreement with biofouling studies for RO systems, considering the key differences between FO and RO. Our findings contradict previous cross-flow studies on particulate/colloidal fouling, where higher cross-flow velocities improved system performance. Thicker spacers reduced the impact of biofouling on FO membrane flux. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Space Station Long Spacer Element begins processing at KSC

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The Long Spacer, a component of the International Space Station, arrives and is moved to its test stand in the northeast corner of the high bay in KSC's Space Station Processing Facility. The Long Spacer provides structural support for the outboard Photovoltaic Modules that supply power to the station. Now just a structure, the Long Spacer will have attached to it as part of processing a heat dissipation radiator and two Pump and Flow Control subassemblies that circulate ammonia to cool the solar array electronics. Also to be mounted are ammonia fluid lines as part of the cooling system and the cabling necessary for power and control of the station. The Long Spacer becomes an integral part of a station truss segment when it is mated with the Integrated Equipment Assembly, which stores the electrical power generated by the solar arrays for use by the station modules. The Long Spacer is being processed in preparation for STS-97, currently planned for launch aboard Discovery in April 1999.

  8. Gas-insulated substation spacer surface degradation analysis

    SciTech Connect

    Chu, F.Y.; Braun, J.M. )

    1990-06-01

    The objective of the project was to develop surface