Menu

Auto Tag: Human

July 7, 2019

Comparative whole genome analysis of three consecutive Salmonella diarizonae isolates.

Infections of very young children or immunocompromised people with Salmonella of higher subspecies are a well-known phenomenon often associated with contact to cold-blooded animals. We describe the molecular characterization of three S. enterica subsp. diarizonae strains, isolated consecutively over a period of several months from a hospital patient suffering from diarrhea and sepsis with fatal outcome. With the initial isolate the first complete genome sequence of a member of subsp. diarizonae is provided and based on this reference we revealed the genomic differences between the three isolates by use of next-generation sequencing and confirmed by phenotypical tests. Genome comparisons revealed mutations within gpt, hfq and purK in the first isolate as a sign of clonal variation rather than host-directed evolution. Furthermore, our work demonstrates that S. enterica subsp. diarizonae possess, besides a conserved set of known Salmonella Pathogenicity Islands, a variable portfolio of additional genomic islands of unknown function. Copyright © 2017 Elsevier GmbH. All rights reserved.

Read more
July 7, 2019

Comparative genomics of maize ear rot pathogens reveals expansion of carbohydrate-active enzymes and secondary metabolism backbone genes in Stenocarpella maydis.

Stenocarpella maydis is a plant pathogenic fungus that causes Diplodia ear rot, one of the most destructive diseases of maize. To date, little information is available regarding the molecular basis of pathogenesis in this organism, in part due to limited genomic resources. In this study, a 54.8 Mb draft genome assembly of S. maydis was obtained with Illumina and PacBio sequencing technologies, and analyzed. Comparative genomic analyses with the predominant maize ear rot pathogens Aspergillus flavus, Fusarium verticillioides, and Fusarium graminearum revealed an expanded set of carbohydrate-active enzymes for cellulose and hemicellulose degradation in S. maydis. Analyses of predicted genes involved in starch degradation revealed six putative a-amylases, four extracellular and two intracellular, and two putative ?-amylases, one of which appears to have been acquired from bacteria via horizontal transfer. Additionally, 87 backbone genes involved in secondary metabolism were identified, which represents one of the largest known assemblages among Pezizomycotina species. Numerous secondary metabolite gene clusters were identified, including two clusters likely involved in the biosynthesis of diplodiatoxin and chaetoglobosins. The draft genome of S. maydis presented here will serve as a useful resource for molecular genetics, functional genomics, and analyses of population diversity in this organism. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Read more
July 7, 2019

Genome architecture and evolution of a unichromosomal asexual nematode.

Asexual reproduction in animals, though rare, is the main or exclusive mode of reproduction in some long-lived lineages. The longevity of asexual clades may be correlated with the maintenance of heterozygosity by mechanisms that rearrange genomes and reduce recombination. Asexual species thus provide an opportunity to gain insight into the relationship between molecular changes, genome architecture, and cellular processes. Here we report the genome sequence of the parthenogenetic nematode Diploscapter pachys with only one chromosome pair. We show that this unichromosomal architecture is shared by a long-lived clade of asexual nematodes closely related to the genetic model organism Caenorhabditis elegans. Analysis of the genome assembly reveals that the unitary chromosome arose through fusion of six ancestral chromosomes, with extensive rearrangement among neighboring regions. Typical nematode telomeres and telomeric protection-encoding genes are lacking. Most regions show significant heterozygosity; homozygosity is largely concentrated to one region and attributed to gene conversion. Cell-biological and molecular evidence is consistent with the absence of key features of meiosis I, including synapsis and recombination. We propose that D. pachys preserves heterozygosity and produces diploid embryos without fertilization through a truncated meiosis. As a prelude to functional studies, we demonstrate that D. pachys is amenable to experimental manipulation by RNA interference. Copyright © 2017 Elsevier Ltd. All rights reserved.

Read more
July 7, 2019

Aestuarium zhoushanense gen. nov., sp. nov., Isolated from the Tidal Flat.

A gram-stain-negative, aerobic, ovoid or short rod-shaped, and non-motile strain, designed G7T was isolated from a tidal flat sample collected from the coast of East Sea in Zhoushan, China. Strain G7T grew at 4-40 °C and pH 6.0-9.0 (optimum, 28 °C and pH 7.5) and with 0-7% (w/v) NaCl (optimum, 1%). The predominant respiratory quinone was Q-10 and the major fatty acids (>10%) identified were C18:1 ?7c, C16:0 and summed feature 3 (C16:1 ?7c and/or C16:1 ?6c). The polar lipids of strain G7T consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, and four unidentified lipids. The genomic DNA G+C content was 56.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain G7T formed a distinct lineage belonging to the Roseobacter clade of the family Rhodobacteraceae. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with the results of phylogenetic analysis, strain G7T is described as a novel species in a new genus, for which the name Aestuarium zhoushanense gen. nov., sp. nov. (type strain G7T = MCCC 1K03229T = KCTC 52584T) is proposed.

Read more
July 7, 2019

Lightweight BWT and LCP merging via the gap algorithm

Recently, Holt and McMillan [Bioinformatics 2014, ACM-BCB 2014] have proposed a simple and elegant algorithm to merge the Burrows-Wheeler transforms of a collection of strings. In this paper we show that their algorithm can be improved so that, in addition to the BWTs, it also merges the Longest Common Prefix (LCP) arrays. Because of its small memory footprint this new algorithm can be used for the final merge of BWT and LCP arrays computed by a faster but memory intensive construction algorithm.

Read more
July 7, 2019

Identification of low allele frequency mosaic mutations in Alzheimer disease

Germline mutations ofAPP,PSEN1, andPSEN2 genes cause autosomal dominant Alzheimer disease (AD). Somatic variants of the same genes may underlie pathogenesis in sporadic AD, which is the most prevalent form of the disease. Importantly, such somatic variants may be present at very low allelic frequency, confined to the brain, and are thus very difficult or impossible to detect in blood-derived DNA. Ever-refined methodologies to identify mutations present in a fraction of the DNA of the original tissue are rapidly transforming our understanding of DNA mutation and their role in complex pathologies such as tumors. These methods stand poised to test to what extend somatic variants may play a role in AD and other neurodegenerative diseases.

Read more
July 7, 2019

Harnessing whole genome sequencing in medical mycology.

Comparative genome sequencing studies of human fungal pathogens enable identification of genes and variants associated with virulence and drug resistance. This review describes current approaches, resources, and advances in applying whole genome sequencing to study clinically important fungal pathogens.Genomes for some important fungal pathogens were only recently assembled, revealing gene family expansions in many species and extreme gene loss in one obligate species. The scale and scope of species sequenced is rapidly expanding, leveraging technological advances to assemble and annotate genomes with higher precision. By using iteratively improved reference assemblies or those generated de novo for new species, recent studies have compared the sequence of isolates representing populations or clinical cohorts. Whole genome approaches provide the resolution necessary for comparison of closely related isolates, for example, in the analysis of outbreaks or sampled across time within a single host.Genomic analysis of fungal pathogens has enabled both basic research and diagnostic studies. The increased scale of sequencing can be applied across populations, and new metagenomic methods allow direct analysis of complex samples.

Read more
July 7, 2019

Complete genetic analysis of a Salmonella enterica serovar Indiana isolate accompanying four plasmids carrying mcr-1, ESBL and other resistance genes in China

One mcr-1-carrying Salmonella enterica serovar Indiana strain D90, was identified from 1320 Salmonella enterica isolates from poultry slaughterhouse in 2012 in China. The objective of this study was to verify the transferability of the mcr-1 gene and also completely characterize the sequence of the strain at the whole-genome level. Broth matting assays were carried out to detect the transferability and whole-genome sequencing (WGS) of S. enterica serovar Indiana D90 was performed using the PacBio RS II system. Open reading frames were assigned using Rapid Annotation using Subsystem Technology (RAST) and analysed by BLASTn and BLASTp. Salmonella Pathogenisity Islands (SPIs) were annotated by SPIFinder platform. The complete genome sequence of S. enterica serovar Indiana D90 contained a circular 4,779,514-bp chromosome and four plasmids. Genome analysis and sequencing revealed that 24 multi-drug resistance (MDR) genes were located on plasmids. The largest plasmid pD90-1, was found to be of an IncHI2/HI2A/Q1/N type that encoded a blaCTX-M-65 gene along with 20 additional antimicrobial resistance genes. A 60.5-kbp IncI2 plasmid pD90-2 contained a nikA-nikB-mcr-1 genetic structure, that can be successfully transferred to E. coli and S. enterica serovar Typhimurium at low transfer rates. Interestingly, comparative sequence analysis revealed the plasmids pD90-1 and pD90-2 showed considerable nucleotide similarity to pHNSHP45-2 and pHNSHP45, respectively. Moreover, the genome and the plasmid pD90-2 also showed high similarity to one carbapenem resistant S. enterica serovar Indiana strain, C629 and its plasmid pC629, respectively. This is the first report of the complete nucleotide sequence of one mcr-1-carrying MDR S. enterica serovar Indiana strain.

Read more
July 7, 2019

Molecular cloning and functional expression of the K(+) channel KV7.1 and the regulatory subunit KCNE1 from equine myocardium.

The voltage-gated K(+)-channel KV7.1 and the subunit KCNE1, encoded by the KCNQ1 and KCNE1 genes, respectively, are responsible for termination of the cardiac action potential. In humans, mutations in these genes can predispose patients to arrhythmias and sudden cardiac death (SCD).To characterize equine KV7.1/KCNE1 currents and compare them to human KV7.1/KCNE1 currents to determine whether KV7.1/KCNE1 plays a similar role in equine and human hearts.mRNA encoding KV7.1 and KCNE1 was isolated from equine hearts, sequenced, and cloned into expression vectors. The channel subunits were heterologously expressed in Xenopus laevis oocytes or CHO-K1 cells and characterized using voltage-clamp techniques.Equine KV7.1/KCNE1 expressed in CHO-K1 cells exhibited electrophysiological properties that are overall similar to the human orthologs; however, a slower deactivation was found which could result in more open channels at fast rates.The results suggest that the equine KV7.1/KCNE1 channel may be important for cardiac repolarization and this could indicate that horses are susceptible to SCD caused by mutations in KCNQ1 and KCNE1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Read more
July 7, 2019

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS(YA), replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Read more
July 7, 2019

Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni.

Campylobacter jejuni, a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms, C. jejuni restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although C. jejuni can be transformed by C. jejuni-derived DNA, it is poorly transformed by the same DNA propagated in Escherichia coli or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term Campylobacter transformation system methyltransferase (ctsM), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a ctsM mutant transforms C. jejuni significantly less well than DNA derived from ctsM(+) (parental) cells. The ctsM mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that E. coli plasmid and PCR-derived DNA can efficiently transform C. jejuni when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform C. jejuni, whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in C. jejuni is distinct from the DNA discrimination described in other competent bacteria.

Read more
July 7, 2019

Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar.

The bacterial enzyme New Delhi metallo-ß-lactamase hydrolyzes almost all ß-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-ß-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1), IncX3 plasmids harboring blaNDM-4 (n = 2) or blaNDM-7 (n = 1), IncFII plasmids harboring blaNDM-4 (n = 1) or blaNDM-5 (n = 3), and a multireplicon F plasmid harboring blaNDM-5 (n = 1). Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

Read more
July 7, 2019

Shared features of cryptic plasmids from environmental and pathogenic Francisella species.

The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We have identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. Genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.

Read more
July 7, 2019

New insights into structural organization and gene duplication in a 1.75-Mb genomic region harboring the a-gliadin gene family in Aegilops tauschii, the source of wheat D genome.

Among the wheat prolamins important for its end-use traits, a-gliadins are the most abundant, and are also a major cause of food-related allergies and intolerances. Previous studies of various wheat species estimated that between 25 and 150 a-gliadin genes reside in the Gli-2 locus regions. To better understand the evolution of this complex gene family, the DNA sequence of a 1.75-Mb genomic region spanning the Gli-2 locus was analyzed in the diploid grass, Aegilops tauschii, the ancestral source of D genome in hexaploid bread wheat. Comparison with orthologous regions from rice, sorghum, and Brachypodium revealed rapid and dynamic changes only occurring to the Ae. tauschii Gli-2 region, including insertions of high numbers of non-syntenic genes and a high rate of tandem gene duplications, the latter of which have given rise to 12 copies of a-gliadin genes clustered within a 550-kb region. Among them, five copies have undergone pseudogenization by various mutation events. Insights into the evolutionary relationship of the duplicated a-gliadin genes were obtained from their genomic organization, transcription patterns, transposable element insertions and phylogenetic analyses. An ancestral glutamate-like receptor (GLR) gene encoding putative amino acid sensor in all four grass species has duplicated only in Ae. tauschii and generated three more copies that are interspersed with the a-gliadin genes. Phylogenetic inference and different gene expression patterns support functional divergence of the Ae. tauschii GLR copies after duplication. Our results suggest that the duplicates of a-gliadin and GLR genes have likely taken different evolutionary paths; conservation for the former and neofunctionalization for the latter.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Read more
July 7, 2019

Avoidance of APOBEC3B-induced mutation by error-free lesion bypass.

APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.