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Sunday, September 22, 2019

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat…

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Sunday, September 22, 2019

Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

DNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1 in vivo leads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes…

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Sunday, September 22, 2019

Acquired interbacterial defense systems protect against interspecies antagonism in the human gut microbiome

The genomes of bacteria derived from the gut microbiota are replete with pathways that mediate contact-dependent interbacterial antagonism. However, the role of direct interactions between co-resident microbes in driving microbiome composition is not well understood. Here we report the widespread occurrence of acquired interbacterial defense (AID) gene clusters in the human gut microbiome. These clusters are found on predicted mobile elements and encode arrays of immunity genes that confer protection against interbacterial toxin-mediated antagonism in vitro and in gnotobiotic mice. We find that Bacteroides ovatus strains containing AID systems that inactivate B. fragilis toxins delivered between cells by the type…

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Sunday, September 22, 2019

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in Borrelia burgdorferi.

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease, Borrelia burgdorferi B31, possesses two RM systems with N6-methyladenosine (m6A) MTase activity, which are encoded by the bbe02 gene located on linear plasmid lp25 and bbq67 on lp56. The specific recognition and/or methylation sequences had not been identified for either of these B. burgdorferi MTases, and it was not previously known whether these RM systems…

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Sunday, September 22, 2019

Whole-genome landscape of Medicago truncatula symbiotic genes.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements…

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Sunday, September 22, 2019

Biparental Inheritance of Mitochondrial DNA in Humans.

Although there has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. Here, we identified three unrelated multigeneration families with a high level of mtDNA heteroplasmy (ranging from 24 to 76%) in a total of 17 individuals. Heteroplasmy of mtDNA was independently examined by high-depth whole mtDNA sequencing analysis in our research laboratory and in two Clinical Laboratory Improvement Amendments and College of American Pathologists-accredited laboratories using multiple approaches. A comprehensive exploration of mtDNA segregation in these families…

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Sunday, September 22, 2019

Regulation of yeast-to-hyphae transition in Yarrowia lipolytica.

The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5′-CCCCT-3′) and upregulation of genes with cell cycle box (5′-ACGCG-3′) motifs in…

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Sunday, September 22, 2019

Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species.

Candida auris is an emergent multidrug-resistant fungal pathogen causing increasing reports of outbreaks. While distantly related to C. albicans and C. glabrata, C. auris is closely related to rarely observed and often multidrug-resistant species from the C. haemulonii clade. Here, we analyze near complete genome assemblies for the four C. auris clades and three related species, and map intra- and inter-species rearrangements across the seven chromosomes. Using RNA-Seq-guided gene predictions, we find that most mating and meiosis genes are conserved and that clades contain either the MTLa or MTLa mating loci. Comparing the genomes of these emerging species to those…

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Sunday, September 22, 2019

Transcriptional landscape of a blaKPC-2 plasmid and response to imipenem exposure in Escherichia coli TOP10.

The diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFIIK plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFIIK-IncFIB blaKPC-2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a…

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Saturday, September 21, 2019

Functional analysis of the first complete genome sequence of a multidrug resistant sequence type 2 Staphylococcus epidermidis.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. The ST2 lineage is frequently multidrug resistant and accounts for most of the clinical disease worldwide. However, there are no publically available, closed ST2 genomes and pathogenesis studies have not focused on these strains. We report the complete genome and methylome of BPH0662, a multidrug resistant, hospital adapted, ST2 S. epidermidis, and describe the correlation between resistome and phenotype, as well as demonstrate its relationship to publically available, international ST2 isolates. Furthermore, we delineate the methylome determined by the two type I restriction modification systems present in BPH0662 through heterologous expression…

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Saturday, September 21, 2019

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7.

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak…

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Saturday, September 21, 2019

Direct detection of DNA methylation during single-molecule, real-time sequencing.

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic…

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Saturday, September 21, 2019

A flexible and efficient template format for circular consensus sequencing and SNP detection.

A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert…

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Saturday, September 21, 2019

The advantages of SMRT sequencing.

Of the current next-generation sequencing technologies, SMRT sequencing is sometimes overlooked. However, attributes such as long reads, modified base detection and high accuracy make SMRT a useful technology and an ideal approach to the complete sequencing of small genomes.

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