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July 7, 2019

Evolution of coreceptor utilization to escape CCR5 antagonist therapy.

The HIV-1 envelope interacts with coreceptors CCR5 and CXCR4 in a dynamic, multi-step process, its molecular details not clearly delineated. Use of CCR5 antagonists results in tropism shift and therapeutic failure. Here we describe a novel approach using full-length patient-derived gp160 quasispecies libraries cloned into HIV-1 molecular clones, their separation based on phenotypic tropism in vitro, and deep sequencing of the resultant variants for structure-function analyses. Analysis of functionally validated envelope sequences from patients who failed CCR5 antagonist therapy revealed determinants strongly associated with coreceptor specificity, especially at the gp120-gp41 and gp41-gp41 interaction surfaces that invite future research on the roles of subunit interaction and envelope trimer stability in coreceptor usage. This study identifies important structure-function relationships in HIV-1 envelope, and demonstrates proof of concept for a new integrated analysis method that facilitates laboratory discovery of resistant mutants to aid in development of other therapeutic agents. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Understanding the genetics of APOE and TOMM40 and role of mitochondrial structure and function in clinical pharmacology of Alzheimer’s disease.

The methodology of Genome-Wide Association Screening (GWAS) has been applied for more than a decade. Translation to clinical utility has been limited, especially in Alzheimer’s Disease (AD). It has become standard practice in the analyses of more than two dozen AD GWAS studies to exclude the apolipoprotein E (APOE) region because of its extraordinary statistical support, unique thus far in complex human diseases. New genes associated with AD are proposed frequently based on SNPs associated with odds ratio (OR) < 1.2. Most of these SNPs are not located within the associated gene exons or introns but are located variable distances away. Often pathologic hypotheses for these genes are presented, with little or no experimental support. By eliminating the analyses of the APOE-TOMM40 linkage disequilibrium region, the relationship and data of several genes that are co-located in that LD region have been largely ignored. Early negative interpretations limited the interest of understanding the genetic data derived from GWAS, particularly regarding the TOMM40 gene. This commentary describes the history and problem(s) in interpretation of the genetic interrogation of the "APOE" region and provides insight into a metabolic mitochondrial basis for the etiology of AD using both APOE and TOMM40 genetics. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Complete nucleotide sequence of pH11, an IncHI2 plasmid conferring multi-antibiotic resistance and multi-heavy metal resistance genes in a clinical Klebsiella pneumoniae isolate.

The complete 284,628bp sequence of pH11, an IncHI2 plasmid, was determined through single-molecule, real-time (SMRT) sequencing. Harbored by a clinical Klebsiella pneumoniae strain H11, and isolated in Beijing, this plasmid contains multiple antibiotic resistance genes, including catA2, aac(6′)-Ib, strB, strA, dfrA19, blaTEM-1, blaSHV-12, sul1, qacE delta 1, ereA, arr2, and aac3. The aac(6′)-Ib is carried by a class I integron. Plasmid pH11 also carries several genes associated with resistance to heavy metals, such as tellurium, mercury, cobalt, zinc, nickel, copper, lead and cadmium. This plasmid exhibits numerous characteristics, including HipBA and RelBE toxin-antitoxin systems, two major transfer (Tra) regions closely related to those of Salmonella enterica serovar plasmid pRH-R27, a type II restriction modification system (EcoRII R-M system), several methyltransferases and methylases and genes encoding Hha and StpA. These characteristics suggest that pH11 may adapt to various hosts and environments. Multiple insertion sequence elements, transposases, recombinases, resolvases and integrases are scattered throughout pH11. The presence of these genes may indicate that horizontal gene transfer occurs frequently in pH11 and thus may facilitate the dissemination of antimicrobial resistance determinants. Our data suggest that pH11 is a chimera gradually assembled through the integration of different horizontally acquired DNA segments via transposition or homologous recombination. Copyright © 2016 Elsevier Inc. All rights reserved.

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July 7, 2019

Regulation of genetic flux between bacteria by restriction-modification systems.

Restriction-modification (R-M) systems are often regarded as bacteria’s innate immune systems, protecting cells from infection by mobile genetic elements (MGEs). Their diversification has been recently associated with the emergence of particularly virulent lineages. However, we have previously found more R-M systems in genomes carrying more MGEs. Furthermore, it has been suggested that R-M systems might favor genetic transfer by producing recombinogenic double-stranded DNA ends. To test whether R-M systems favor or disfavor genetic exchanges, we analyzed their frequency with respect to the inferred events of homologous recombination and horizontal gene transfer within 79 bacterial species. Genetic exchanges were more frequent in bacteria with larger genomes and in those encoding more R-M systems. We created a recognition target motif predictor for Type II R-M systems that identifies genomes encoding systems with similar restriction sites. We found more genetic exchanges between these genomes, independently of their evolutionary distance. Our results reconcile previous studies by showing that R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems. Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.

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July 7, 2019

Isolation and complete genome sequence of the thermophilic Geobacillus sp. 12AMOR1 from an Arctic deep-sea hydrothermal vent site.

Members of the genus Geobacillus have been isolated from a wide variety of habitats worldwide and are the subject for targeted enzyme utilization in various industrial applications. Here we report the isolation and complete genome sequence of the thermophilic starch-degrading Geobacillus sp. 12AMOR1. The strain 12AMOR1 was isolated from deep-sea hot sediment at the Jan Mayen hydrothermal Vent Site. Geobacillus sp. 12AMOR1 consists of a 3,410,035 bp circular chromosome and a 32,689 bp plasmid with a G?+?C content of 52 % and 47 %, respectively. The genome comprises 3323 protein-coding genes, 88 tRNA species and 10 rRNA operons. The isolate grows on a suite of sugars, complex polysaccharides and proteinous carbon sources. Accordingly, a versatility of genes encoding carbohydrate-active enzymes (CAZy) and peptidases were identified in the genome. Expression, purification and characterization of an enzyme of the glycoside hydrolase family 13 revealed a starch-degrading capacity and high thermal stability with a melting temperature of 76.4 °C. Altogether, the data obtained point to a new isolate from a marine hydrothermal vent with a large bioprospecting potential.

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July 7, 2019

Genomic organization of the zebrafish (Danio rerio) T cell receptor alpha/delta locus and analysis of expressed products.

In testing the hypothesis that all jawed vertebrate classes employ immunoglobulin heavy chain V (IgHV) gene segments in their T cell receptor (TCR)d encoding loci, we found that some basic characterization was required of zebrafish TCRd. We began by annotating and characterizing the TCRa/d locus of Danio rerio based on the most recent genome assembly, GRCz10. We identified a total of 141 theoretically functional V segments which we grouped into 41 families based upon 70 % nucleotide identity. This number represents the second greatest count of apparently functional V genes thus far described in an antigen receptor locus with the exception of cattle TCRa/d. Cloning, relative quantitative PCR, and deep sequencing results corroborate that zebrafish do express TCRd, but these data suggest only at extremely low levels and in limited diversity in the spleens of the adult fish. While we found no evidence for IgH-TCRd rearrangements in this fish, by determining the locus organization we were able to suggest how the evolution of the teleost a/d locus could have lost IgHVs that exist in sharks and frogs. We also found evidence of surprisingly low TCRd expression and repertoire diversity in this species.

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July 7, 2019

Genetic diversity of O-antigens in Hafnia alvei and the development of a suspension array for serotype detection.

Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.

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July 7, 2019

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n?=?14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

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July 7, 2019

Population scale mapping of transposable element diversity reveals links to gene regulation and epigenomic variation.

Variation in the presence or absence of transposable elements (TEs) is a major source of genetic variation between individuals. Here, we identified 23,095 TE presence/absence variants between 216 Arabidopsis accessions. Most TE variants were rare, and we find these rare variants associated with local extremes of gene expression and DNA methylation levels within the population. Of the common alleles identified, two thirds were not in linkage disequilibrium with nearby SNPs, implicating these variants as a source of novel genetic diversity. Many common TE variants were associated with significantly altered expression of nearby genes, and a major fraction of inter-accession DNA methylation differences were associated with nearby TE insertions. Overall, this demonstrates that TE variants are a rich source of genetic diversity that likely plays an important role in facilitating epigenomic and transcriptional differences between individuals, and indicates a strong genetic basis for epigenetic variation.

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July 7, 2019

Resurgence of less-studied smut fungi as models of phytopathogenesis in the -omics era.

The smut fungi form a large, diverse, and non-monophyletic group of plant pathogens that have long served as both important pests of human agriculture but also as fertile organisms of scientific investigation. As modern techniques of molecular genetic analysis became available, many previously-studied species that proved refractive to these techniques fell by the wayside to become neglected. Now, as the advent of rapid and affordable next-generation sequencing provides genomic and transcriptomic resources for even these “forgotten” fungi, several species are making a come-back and retaking prominent places in phytopathogenic research. In this review, we highlight several of these smut fungi, with special emphasis on Microbotryum lychnidis-dioicae, an anther smut, whose molecular genetic tools have finally begun to catch up with its historical importance in classical genetics and now provide mechanistic insights for ecological studies, evolution of host/pathogen interaction, and investigations of emerging infectious disease.

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July 7, 2019

ABO allele-level frequency estimation based on population-scale genotyping by next generation sequencing.

The characterization of the ABO blood group status is vital for blood transfusion and solid organ transplantation. Several methods for the molecular characterization of the ABO gene, which encodes the alleles that give rise to the different ABO blood groups, have been described. However, the application of those methods has so far been restricted to selected samples and not been applied to population-scale analysis.We describe a cost-effective method for high-throughput genotyping of the ABO system by next generation sequencing. Sample specific barcodes and sequencing adaptors are introduced during PCR, rendering the products suitable for direct sequencing on Illumina MiSeq or HiSeq instruments. Complete sequence coverage of exons 6 and 7 enables molecular discrimination of the ABO subgroups and many alleles. The workflow was applied to ABO genotype more than a million samples. We report the allele group frequencies calculated on a subset of more than 110,000 sampled individuals of German origin. Further we discuss the potential of the workflow for high resolution genotyping taking the observed allele group frequencies into account. Finally, sequence analysis revealed 287 distinct so far not described alleles of which the most abundant one was identified in 174 samples.The described workflow delivers high resolution ABO genotyping at low cost enabling population-scale molecular ABO characterization.

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July 7, 2019

A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer.

Although human LINE-1 (L1) elements are actively mobilized in many cancers, a role for somatic L1 retrotransposition in tumor initiation has not been conclusively demonstrated. Here, we identify a novel somatic L1 insertion in the APC tumor suppressor gene that provided us with a unique opportunity to determine whether such insertions can actually initiate colorectal cancer (CRC), and if so, how this might occur. Our data support a model whereby a hot L1 source element on Chromosome 17 of the patient’s genome evaded somatic repression in normal colon tissues and thereby initiated CRC by mutating the APC gene. This insertion worked together with a point mutation in the second APC allele to initiate tumorigenesis through the classic two-hit CRC pathway. We also show that L1 source profiles vary considerably depending on the ancestry of an individual, and that population-specific hot L1 elements represent a novel form of cancer risk. © 2016 Scott et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Selfish drive can trump function when animal mitochondrial genomes compete.

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes showed that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection, promoting change in the sequences influencing transmission.

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July 7, 2019

Resistance from relatives.

Crops are made resistant to pathogens such as wheat stem rust, Asian soybean rust and potato late blight by methods to access the pool of resistance genes present in related plants.

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July 7, 2019

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic.

Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors. © 2016 Verfaillie et al.; Published by Cold Spring Harbor Laboratory Press.

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