12 research outputs found

    SCA10 expansion templates for SMRT sequencing.

    No full text
    <p>(A) PCR amplification of the SCA10 expansion from gDNA extracted from blood lymphocytes (Subjects A and B) or from somatic cell hybrid lines (subject C). Lanes are cropped from non-adjacent lanes of the same gel. The full gel is shown is <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.s001" target="_blank">S1 Fig</a>. Arrows indicate the size of bands that were excised for cloning and sequencing (subject A, the 6.5 kb band; subject B, the 5.9 kb band; subject C, the 4.7 kb band) (B) Purified template from cloned PCR products in Fig 1A for SMRT sequencing. L: 1 kb ladder (New England Biolabs). The full gels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.s002" target="_blank">S2 Fig</a>.</p

    pJAZZ-OCmin vector.

    No full text
    <p>The telN gene and several non-essential genes from phage N15 (purple boxes) were deleted from the vector pJAZZ-OC. repA, cB: replication protein genes; Cam<sup>r</sup>, chloramphenicol resistance; T, terminator. Closed terminal hairpin structures are indicated by black circles.</p

    Schematic representations of the repeat expansions.

    No full text
    <p>(A) SCA10 expansion in subject A. (B) SCA10 expansion in subject B. (C) SCA10 expansion in subject C. Rectangles represent sequence motifs, as indicated by the color key, in the 5’ (upper left) to 3’ (lower right) direction. Black rectangles indicate unverified motifs described further in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.g003" target="_blank">Fig 3B</a> and are indicated as follows: A: ATTTCT, ATTTCT; B: ATTTCT, A, ATTTCT, A, ATTCCT, TAC, ATTTCT, A, ATT, ACTTCT, ATTCA, ATTTCT, ATTTCT, T, ACTTTCT, TCTTTCT, ATTT, ATTTCT, ATCT, ATTTCT, ATTTCT, ATTTCT, ATTTCT, ATTTCT, T, ATCC, ATTC, ATTTCC, C, ATTTCC, TTCCC, ATTTCC, CATCC, ATTTCC, C, C, C, C, ATTC, ATTTCC, ATTCC; C: ATCT, ATCT, ATCT, AT, ATCT, T, ATC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, C, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, C</p

    Proportion of repeat motifs in SCA10 expansions.

    No full text
    <p>Proportions are calculated as the percentage of nucleotides of each motifs divided by the total number of nucleotides for each expansion. Motifs present in SMRT sequence results that are verified by Sanger sequencing methods (“shotgun”) comprise the majority of motifs seen (A) while some motifs are unverified (B). Green, SMRT sequencing results from subject A; Green hatched, random shotgun sequencing results from subject A; Blue, SMRT sequencing results from subject B; Red, SMRT sequencing result from subject C; Red hatched, random shotgun sequencing results from subject C.</p

    SMRT sequencing results summary.

    No full text
    <p><sup>§</sup>as estimated by gel electrophoresis of cloned expansion fragment excised from plasmid backbone.</p><p><sup><b>¶</b></sup><b>As compared with Sanger sequencing.</b></p><p>*Alignment based on reference genome sequence (NC_000022.11; GI: 568815576, Region: 45794523..45796589) using LALIGN.</p><p>^as determined by counting motif blocks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.g002" target="_blank">Fig 2</a>. Rpts, repeats; nts, nucleotides; bp, base pairs</p><p>SMRT sequencing results summary.</p

    Components of the nitrogen metabolism of <i>Ca</i>. N. evergladensis

    No full text
    <p>: ammonia oxidation (4, 5), ammonia assimilation (8, 9, 10), nitrite reduction (6), nitrous oxide production (7). Reactions are mediated by the following transporters and enzymes: urea transporters, urease (1, 2), ammonia transporters (3), archaeal ammonia monooxygenase (AMO) (4), candidate enzyme: multicopper oxidase (5), nitrite reductase (NirK) (6), nitric oxide reductase (NorD, NorQ), catalytic subunit (NorB) is missing (7), glutamate dehydrogenase (8), glutamine synthetase (9), glutamate synthase (10). NO may upregulate activity of AMO. * - experimental evidences are needed.</p

    Comparison of protein coding sequences (CDS) of <i>Ca</i>. Nitrososphaera evergladensis with CDS of other ammonia-oxidizing archaea.

    No full text
    <p>(A) CDS of <i>Ca</i>. Nitrososphaera evergladensis were compared to CDS of <i>Ca</i>. N. gargensis. (B) CDS of the group I.1a (<i>N. maritimus</i>, <i>Ca</i>. N. sediminis, <i>C. symbiosum</i>, <i>Ca</i>. N. limnia, <i>Ca</i>. N. koreensis) were compared to CDS of the group I.1b (<i>Ca</i>. N. evergladensis and <i>Ca</i>. N. gargensis). Overlapping regions represent CDS with amino acid sequence identity 35% and higher.</p

    A phylogenetic tree of ammonia-oxidizing archaea 16S rRNA gene sequences (about 1.4 kb).

    No full text
    <p>23 16S rRNA sequences of AOA were randomly selected from the National Center for Biotechnology Information databases. Conservative sites (1.08 kb) were selected using Gblocks. The branching patterns in the maximum-likelihood tree are denoted by their respective bootstrap values (1000 iterations).</p

    Genome Sequence of <i>Candidatus</i> Nitrososphaera evergladensis from Group I.1b Enriched from Everglades Soil Reveals Novel Genomic Features of the Ammonia-Oxidizing Archaea

    No full text
    <div><p>The activity of ammonia-oxidizing archaea (AOA) leads to the loss of nitrogen from soil, pollution of water sources and elevated emissions of greenhouse gas. To date, eight AOA genomes are available in the public databases, seven are from the group I.1a of the Thaumarchaeota and only one is from the group I.1b, isolated from hot springs. Many soils are dominated by AOA from the group I.1b, but the genomes of soil representatives of this group have not been sequenced and functionally characterized. The lack of knowledge of metabolic pathways of soil AOA presents a critical gap in understanding their role in biogeochemical cycles. Here, we describe the first complete genome of soil archaeon <i>Candidatus</i> Nitrososphaera evergladensis, which has been reconstructed from metagenomic sequencing of a highly enriched culture obtained from an agricultural soil. The AOA enrichment was sequenced with the high throughput next generation sequencing platforms from Pacific Biosciences and Ion Torrent. The <i>de novo</i> assembly of sequences resulted in one 2.95 Mb contig. Annotation of the reconstructed genome revealed many similarities of the basic metabolism with the rest of sequenced AOA. <i>Ca</i>. N. evergladensis belongs to the group I.1b and shares only 40% of whole-genome homology with the closest sequenced relative <i>Ca</i>. N. gargensis. Detailed analysis of the genome revealed coding sequences that were completely absent from the group I.1a. These unique sequences code for proteins involved in control of DNA integrity, transporters, two-component systems and versatile CRISPR defense system. Notably, genomes from the group I.1b have more gene duplications compared to the genomes from the group I.1a. We suggest that the presence of these unique genes and gene duplications may be associated with the environmental versatility of this group.</p></div
    corecore