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Scientific publications carry a practical moral duty: they must last. Along that line of thinking, some methods are proposed to allow economically and structurally viable publication of DNA sequence data of any size in printed matter and PDFs. The proposal is primarily aimed at contributing for preserving information for the future, while allowing authors to avoid information splitting and complement storage ex situ, that is, in server machines, outside the publication proper. The technique may also help to solve the impasse between the ICZN Code requirement that a new nomen be associated to diagnostic characters for the taxon vs. the phylogenetic definition of taxa, based on cladograms only: sequence data are characters, and can now be easily and comfortably included in taxonomic publications, with direct textual mention to their diagnostic sections. The compression level achieved allows the inclusion of all wanted DNA or RNA sequences in the same printed matter or PDF publications where the sequences are cited and discussed. Reduced font sizes, invisible fonts, and original 2D black & white and color barcodes are illustrated and briefly discussed. The level of data compression achieved can allow each full page of sequence data, or about 5000 characters, to be precisely coded into a color barcode as small as a square of 1.5 mm. A practical example is provided with Taeniogonalos woodorum Smith (Hymenoptera, Trigonalidae). Free software to generate publishable barcodes from txt or FASTA files is provided at www.systaxon.ufes.br/dna.

In this paper we (1) introduce a directed graphical representation of DNA primary sequences; (2) describe a scheme that transforms the directed graph of a DNA sequence into an upper triangular matrix; (3) investigate whether or not the existing matrix-based invariants of DNA sequences are compatible for the upper triangular matrix representation. The utility of our method is illustrated by an examination of the similarity between human and other seven species.

The uncoupling proteins (UCPs) are mitochondrial transporters that modulate the efficiency of oxidative phosphorylation. Members of this family have been described in many phyla within the animal and plant kingdoms, as well as in fungi. The mammalian uncoupling protein UCP1 is activated by fatty acids and inhibited by nucleotides. In the absence of both regulators, UCP1 presents a high ohmic proton conductance that is a unique property of this carrier. The increasing number of protein sequences available has enabled us to apply a sequence analysis approach to investigate transporter function. We reconstructed a robust phylogeny of UCPs and used comparative sequence analysis to search for phylogenetically shared derived sequence features that may confer distinct properties on UCP1. We assessed the functional relevance of shared derived UCP1 residues by substituting them with their counterparts in UCP2, and expressing the protein chimeras in yeast. We found that substitution of both Glu134 and Met140 abolishes the basal proton permeability of UCP1 while preserving fatty acid activation and its nucleotide inhibition.

The first problem to be solved is to map DNA sequences onto numerical sequences in bio molecular sequence analysis by mathematical, physical and digital signal processing methods. The characters and the adaptability of eight existing mapping methods are analyzed in this paper. A new numerical mapping method based on the probability of bases in the segment DNA sequence is presented. Most of the coding sequences are characterized by 3 - base periodicity. Further more, eight numerical mapping methods are compared and the new method is verified by means of the spectrum analysis of DNA sequence with 3 - base periodicity. The computer simulation results show that the mapping method based complex plane is superior to the other seven methods in reflecting the original information of the bio-molecular sequence and the quality of the obtained power spectra. The identification rate the new method attains is approximately what the complex plane method has achieved.

In this paper, we proposed a new type of seed for Blast-like homology search tools called "half seed''. This new seed is better than the "consecutive seed'' used by the original Blast tools in both sensitivity and efficiency. When compared with the "gapped seed'', which is proposed together with a new Blast-like searching tool, PatternHunter, this new seed offers a much wider range of choices for performing tradeoff between sensitivity and efficiency. This property is especially useful when some searching applications want to get more precise results with limitation on hardware resources, or vice versa.

We present the novel HLA-allele B*2722 amplified and sequenced from a Filipino individual. This DNA was included several times in the International Cell Exchange, UCLA, and typed at the time as B*27 or B*2706 by the majority of the participating laboratories. The second HLA-B allele of this person is B*3802. B*2704/06 or B*2704/06/10 were suggested by approximately one-third of the laboratories. As we were investigating the intron 3 sequences of B*27 alleles, we also sequenced exon 4 of this Filipino DNA and found a single nucleotide exchange in exon 4 (pos. 704) which was not in concordance with the previous B*2706 typing. Our sequencing results showed that the coding sequence of B*2722 is identical to B*2706 in exon 1, 2 and 3. In exon 4 the B*2722 sequence is identical to B*2704. HLA-B*2704 differs from B*2706 in one base at position 704, a G in B*2704 and a C in B*2706, respectively.

We describe a number of improvements to the CAP sequence assembly program. These improvements include the development of methods for solving the problem caused by simple repetitive sequences, for automatically editing fragment alignments and consensus sequences, and for identifying chimeric fragments. The improved program (CAP2) assembled each of seven data sets, six of which contain repetitive sequences of very strong similarity, into a single sequence. As an example, CAP2 assembled a set of 1467 fragments into a single sequence of 73,328 bp that has only eight differences from the original sequence. The effects of fragment length, coverage, and error rate on the performance of CAP2 were evaluated using artificial data sets.

We have developed a World Wide Web (WWW) version of the sequence retrieval system Query: WWW-Query. This server allows to query nucleotide sequence banks in the EMBL/GenBank/DDBJ formats and protein sequence banks in the NBRF/PIR format. WWW-Query includes all the features of the on-line sequences browsers already available: possibility to build complex queries, integration of cross-references with different data banks, and access to the functional zones of biological interest. It also provides original services not available elsewhere: introduction of the notion of re-usable sequence lists, integration of dedicated helper applications for visualizing alignments and phylogenetic trees and links with multivariate methods for studying codon usage or for complementing phylogenies.

The evolutionary conserved region of the equine homologue of the p53 gene from the donkey genome was PCR amplified and cloned. The 1380 bp fragment consisted of exons 5 to 9 and the intervening introns. The exonic and intronic DNA sequences showed a variable but high level of homology with previously published human sequences. The aminoacid sequences corresponding to the evolutionary conserved domains II, III, and V were identical to the human regions, whilst domain IV was 96% homologous.

We describe evidence that DNA sequences from vectors used for cloning and sequencing have been incorporated accidentally into eukaryotic entries in the GenBank database. These incorporations were not restricted to one type of vector or to a single mechanism. Many minor instances may have been the result of simple editing errors, but some entries contained large blocks of vector sequence that had been incorporated by contamination or other accidents during cloning. Some cases involved unusual rearrangements and areas of vector distant from the normal insertion sites. Matches to vector were found in 0.23% of 20,000 sequences analyzed in GenBank Release 63. Although the possibility of anomalous sequence incorporation has been recognized since the inception of GenBank and should be easy to avoid, recent evidence suggests that this problem is increasing more quickly than the database itself. The presence of anomalous sequence may have serious consequences for the interpretation and use of database entries, and will have an impact on issues of database management. The incorporated vector fragments described here may also be useful for a crude estimate of the fidelity of sequence information in the database. In alignments with well-defined ends, the matching sequences showed 96.8% identity to vector; when poorer matches with arbitrary limits were included, the aggregate identity to vector sequence was 94.8%.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

The type strain SUR2 of the novel species Chryseobacterium limigenitum was isolated from a dehydrated sludge of the municipal sewage treatment plant in Dogoše near Maribor in Slovenia. The draft genome, with 60 contigs, 4,697,725bp, 34.4% of G+C content, was obtained using the Illumina HiSeq 2500-1 platform. Joint Genome Institute Microbial Genome Annotation Pipeline (MGAP v.4) has identified 4322 protein-coding sequences including resistance genes against arsenic and other heavy metals. In addition, a subclass B3 metallo-β-lactamase, which confers resistance to penicillins, cephalosporins and carbapenems, was also present in the genome. The genome sequence provides important information regarding bioremediation potential and pathogenic properties of this newly identified species.

BackgroundCongenital contractural arachnodactyly (CCA) is an autosomal dominant rare genetic disease, estimated to be less than 1 in 10,000 worldwide. People with this condition often have permanently bent joints (contractures), like bent fingers and toes (camptodactyly).Case presentationIn this study, we investigated the genetic aetiology of CCA in a four-generation Chinese family. The blood samples were collected from 22 living members of the family in the Yangquan County, Shanxi Province, China. Of those, eight individuals across 3 generations have CCA. Whole exome sequencing (WES) identified a missense mutation involving a T-to-G transition at position 3229 (c.3229 T > G) in exon 25 of the FBN2 gene, resulting in a Cys 1077 to Gly change (p.C1077G). This previously unreported mutation was found in all 8 affected individuals, but absent in 14 unaffected family members. SIFT/PolyPhen prediction and protein conservation analysis suggest that this novel mutation is pathogenic. Our study extended causative mutation spectrum of FBN2 gene in CCA patients.ConclusionsThis study has identified a novel missense mutation in FBN2 gene (p.C1077G) resulting in CCA in a family of China.

Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.

One of the first protein polymorphisms identified in humans involves the abundant blood protein haptoglobin. Two exons of the HP gene (encoding haptoglobin) exhibit copy number variation that affects HP protein structure and multimerization. The evolutionary origins and medical relevance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from many recurring deletions, more specifically, reversions of an ancient hominin-specific duplication of these exons. Although this polymorphism has been largely invisible to genome-wide genetic studies thus far, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We further show that these deletions, and a SNP that affects HP expression, appear to drive the strong association of cholesterol levels with SNPs near HP. Recurring exonic deletions in HP likely enhance human health by lowering cholesterol levels in the blood.

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key rate-limiting step in HIV-1 transcription and latency reversal. The viral Tat protein recruits human super elongation complexes (SECs) to paused Pol II to overcome this restriction. Despite the recent progress in understanding the functions of different subsets of SECs in controlling cellular and Tat-activated HIV transcription, little is known about the SEC subtypes that help reverse viral latency in CD4(+) T cells. Here, we used the CRISPR-Cas9 genome-editing tool to knock out the gene encoding the SEC subunit ELL2, AFF1, or AFF4 in Jurkat/2D10 cells, a well-characterized HIV-1 latency model. Depletion of these proteins drastically reduced spontaneous and drug-induced latency reversal by suppressing HIV-1 transcriptional elongation. Surprisingly, a low-abundance subset of SECs containing ELL2 and AFF1 was found to play a predominant role in cooperating with Tat to reverse latency. By increasing the cellular level/activity of these Tat-friendly SECs, we could potently activate latent HIV-1 without using any drugs. These results implicate the ELL2/AFF1-SECs as an important target in the future design of a combinatorial therapeutic approach to purge latent HIV-1.

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans.

Alternative splicing (AS) is a robust generator of mammalian transcriptome complexity. Splice site specification is controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. These interactions are frequently localized to the intronic U-rich polypyrimidine tracts (PPT) located 5' to the majority of splice acceptor junctions. αCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNPEs) comprise a subset of KH-domain proteins with high affinity and specificity for C-rich polypyrimidine motifs. Here, we demonstrate that αCPs promote the splicing of a defined subset of cassette exons via binding to a C-rich subset of polypyrimidine tracts located 5' to the αCP-enhanced exonic segments. This enhancement of splice acceptor activity is linked to interactions of αCPs with the U2 snRNP complex and may be mediated by cooperative interactions with the canonical polypyrimidine tract binding protein, U2AF65. Analysis of αCP-targeted exons predicts a substantial impact on fundamental cell functions. These findings lead us to conclude that the αCPs play a direct and global role in modulating the splicing activity and inclusion of an array of cassette exons, thus driving a novel pathway of splice site regulation within the mammalian transcriptome.

4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.

The complete mitochondrial genome was sequenced in two individuals of Sakhalin taimen Parahucho perryi. The genome sizes were 16,652 bp and 16,653 bp in the two isolates. Gene arrangement, base composition, and size of the two sequenced haplotypes are very similar to the P. perryi genome previously published (KC897653), but do not contain two frame-disrupting indels in the ND4 gene.

The complete mitochondrial genomes were sequenced from the Northern and Southern Dolly Varden chars, Salvelinus malma and S. curilus. The genome sequences are 16,654 bp in size in both species, and the gene arrangement, composition, and size are very similar to the salmonid fish genomes published previously. The level of sequence divergence between S. malma and S. curilus inferred from the complete mitochondrial genomes is relatively low (1.88%) indicating recent divergence of the species and/or historical hybridization.

The complete mitochondrial genomes were sequenced in two individuals of Siberian taimen Hucho taimen. The sizes of the genomes were 16,833 and 16,914 in the two isolates, representing two haplotype groups previously detected. The gene arrangement, base composition, and size of the two sequenced genomes are very similar to the H. taimen genome previously published (HQ897271), but did not contain introgressed fragments from two subspecies of lenok, Brachymystax lenok and B. l. tsinlingensis.

Robinow syndrome is a rare congenital disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features. Recent reports have identified, in individuals with dominant Robinow syndrome, a specific type of variant characterized by being uniformly located in the penultimate exon of DVL1 and resulting in a -1 frameshift allele with a premature termination codon that escapes nonsense-mediated decay. Here, we studied a cohort of individuals who had been clinically diagnosed with Robinow syndrome but who had not received a molecular diagnosis from variant studies of DVL1, WNT5A, and ROR2. Because of the uniform location of frameshift variants in DVL1-mediated Robinow syndrome and the functional redundancy of DVL1, DVL2, and DVL3, we elected to pursue direct Sanger sequencing of the penultimate exon of DVL1 and its paralogs DVL2 and DVL3 to search for potential disease-associated variants. Remarkably, targeted sequencing identified five unrelated individuals harboring heterozygous, de novo frameshift variants in DVL3, including two splice acceptor mutations and three 1 bp deletions. Similar to the variants observed in DVL1-mediated Robinow syndrome, all variants in DVL3 result in a -1 frameshift, indicating that these highly specific alterations might be a common cause of dominant Robinow syndrome. Here, we review the current knowledge of these peculiar variant alleles in DVL1- and DVL3-mediated Robinow syndrome and further elucidate the phenotypic features present in subjects with DVL1 and DVL3 frameshift mutations.

BackgroundIn congenital Factor (F) VII deficiency bleeding phenotype and intrinsic FVII activity levels don't always correlate. Patients with FVII activity levels

Robinow syndrome is a rare congenital disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features. Recent reports have identified, in individuals with dominant Robinow syndrome, a specific type of variant characterized by being uniformly located in the penultimate exon of DVL1 and resulting in a -1 frameshift allele with a premature termination codon that escapes nonsense-mediated decay. Here, we studied a cohort of individuals who had been clinically diagnosed with Robinow syndrome but who had not received a molecular diagnosis from variant studies of DVL1, WNT5A, and ROR2. Because of the uniform location of frameshift variants in DVL1-mediated Robinow syndrome and the functional redundancy of DVL1, DVL2, and DVL3, we elected to pursue direct Sanger sequencing of the penultimate exon of DVL1 and its paralogs DVL2 and DVL3 to search for potential disease-associated variants. Remarkably, targeted sequencing identified five unrelated individuals harboring heterozygous, de novo frameshift variants in DVL3, including two splice acceptor mutations and three 1 bp deletions. Similar to the variants observed in DVL1-mediated Robinow syndrome, all variants in DVL3 result in a -1 frameshift, indicating that these highly specific alterations might be a common cause of dominant Robinow syndrome. Here, we review the current knowledge of these peculiar variant alleles in DVL1- and DVL3-mediated Robinow syndrome and further elucidate the phenotypic features present in subjects with DVL1 and DVL3 frameshift mutations.

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box-like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met.

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients' combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70-associated autoimmune disease.

Crocosphaera watsonii is a unicellular nitrogen (N2)-fixing cyanobacterium with ecological importance in oligotrophic oceans. In cultivated strains there are two phenotypes of C. watsonii (large and small cells) with differences that could differentially impact biogeochemical processes. Recent work has shown the phenotypes diverged through loss or addition of type-specific genes in a fraction of their genomes, whereas the rest of the genomes were maintained at 99-100% DNA identity. Previous molecular assays for C. watsonii abundances targeted the conserved regions and therefore could not differentiate between phenotypes, so their relative distributions in natural communities were unknown. To determine phenotype distributions, this study developed and applied type-specific quantitative polymerase chain reaction assays to samples from the North and South Pacific. Abundances of both Crocosphaera types declined sharply with depth between 45 and 75 m in both sites. In surface water small cells were 10-100 times more abundant than large cells in the N. Pacific, whereas in the S. Pacific the two phenotypes were nearly equal. Evidence for large cell aggregation was only found in N. Pacific samples. The differences in C. watsonii sub-populations in the North and South Pacific have direct implications for biogeochemistry and carbon export in oligotrophic gyres.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL in vivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

MicroRNAs have been associated with many different biological functions, but little is known about their roles in conditioned behavior. We demonstrate that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability were both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicated that miR-980 regulates neuronal excitability. We identified the autism susceptibility gene, A2bp1, as an mRNA target for miR-980. A2bp1 levels varied inversely with miR-980 expression; memory performance was directly related to A2bp1 levels. In addition, A2bp1 knockdown reversed the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. Our results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement.

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling.

Crocosphaera watsonii is a unicellular nitrogen (N2)-fixing cyanobacterium with ecological importance in oligotrophic oceans. In cultivated strains there are two phenotypes of C. watsonii (large and small cells) with differences that could differentially impact biogeochemical processes. Recent work has shown the phenotypes diverged through loss or addition of type-specific genes in a fraction of their genomes, whereas the rest of the genomes were maintained at 99-100% DNA identity. Previous molecular assays for C. watsonii abundances targeted the conserved regions and therefore could not differentiate between phenotypes, so their relative distributions in natural communities were unknown. To determine phenotype distributions, this study developed and applied type-specific quantitative polymerase chain reaction assays to samples from the North and South Pacific. Abundances of both Crocosphaera types declined sharply with depth between 45 and 75 m in both sites. In surface water small cells were 10-100 times more abundant than large cells in the N. Pacific, whereas in the S. Pacific the two phenotypes were nearly equal. Evidence for large cell aggregation was only found in N. Pacific samples. The differences in C. watsonii sub-populations in the North and South Pacific have direct implications for biogeochemistry and carbon export in oligotrophic gyres.

A major unanswered question in biology and medicine is the mechanism by which the product of the apolipoprotein E ε4 allele, the lipid-binding protein apolipoprotein E4 (ApoE4), plays a pivotal role in processes as disparate as Alzheimer's disease (AD; in which it is the single most important genetic risk factor), atherosclerotic cardiovascular disease, Lewy body dementia, hominid evolution, and inflammation. Using a combination of neural cell lines, skin fibroblasts from AD patients, and ApoE targeted replacement mouse brains, we show in the present report that ApoE4 undergoes nuclear translocation, binds double-stranded DNA with high affinity (low nanomolar), and functions as a transcription factor. Using chromatin immunoprecipitation and high-throughput DNA sequencing, our results indicate that the ApoE4 DNA binding sites include ∼1700 gene promoter regions. The genes associated with these promoters provide new insight into the mechanism by which AD risk is conferred by ApoE4, because they include genes associated with trophic support, programmed cell death, microtubule disassembly, synaptic function, aging, and insulin resistance, all processes that have been implicated in AD pathogenesis. Significance statement: This study shows for the first time that apolipoprotein E4 binds DNA with high affinity and that its binding sites include 1700 promoter regions that include genes associated with neurotrophins, programmed cell death, synaptic function, sirtuins and aging, and insulin resistance, all processes that have been implicated in Alzheimer's disease pathogenesis.

Based on chromosome sequences, the human pathogen Borrelia miyamotoi phylogenetically clusters with species that cause relapsing fever. But atypically for relapsing fever agents, B. miyamotoi is transmitted not by soft ticks but by hard ticks, which also are vectors of Lyme disease Borrelia species. To further assess the relationships of B. miyamotoi to species that cause relapsing fever, I investigated extrachromosomal sequences of a North American strain with specific attention on plasmid-borne vsp and vlp genes, which are the underpinnings of antigenic variation during relapsing fever. For a hybrid approach to achieve assemblies that spanned more than one of the paralogous vsp and vlp genes, a database of short-reads from next-generation sequencing was supplemented with long-reads obtained with real-time DNA sequencing from single polymerase molecules. This yielded three contigs of 31, 16, and 11 kb, which each contained multiple and diverse sequences that were homologous to vsp and vlp genes of the relapsing fever agent B. hermsii. Two plasmid fragments had coding sequences for plasmid partition proteins that differed from each other from paralogous proteins for the megaplasmid and a small plasmid of B. miyamotoi. One of 4 vsp genes, vsp1, was present at two loci, one of which was downstream of a candiate prokaryotic promoter. A limited RNA-seq analysis of a population growing in the blood of mice indicated that of the 4 different vsp genes vsp1 was the one that was expressed. The findings indicate that B. miyamotoi has at least four types of plasmids, two or more of which bear vsp and vlp gene sequences that are as numerous and diverse as those of relapsing fever Borrelia. The database and insights from these findings provide a foundation for further investigations of the immune responses to this pathogen and of the capability of B. miyamotoi for antigenic variation.

Single amplified genomes and genomes assembled from metagenomes have enabled the exploration of uncultured microorganisms at an unprecedented scale. However, both these types of products are plagued by contamination. Since these genomes are now being generated in a high-throughput manner and sequences from them are propagating into public databases to drive novel scientific discoveries, rigorous quality controls and decontamination protocols are urgently needed. Here, we present ProDeGe (Protocol for fully automated Decontamination of Genomes), the first computational protocol for fully automated decontamination of draft genomes. ProDeGe classifies sequences into two classes--clean and contaminant--using a combination of homology and feature-based methodologies. On average, 84% of sequence from the non-target organism is removed from the data set (specificity) and 84% of the sequence from the target organism is retained (sensitivity). The procedure operates successfully at a rate of ~0.30 CPU core hours per megabase of sequence and can be applied to any type of genome sequence.

Adipose tissue plays important roles in regulating carbohydrate and lipid homeostasis, but less is known about the regulation of amino acid metabolism in adipocytes. Here we applied isotope tracing to pre-adipocytes and differentiated adipocytes to quantify the contributions of different substrates to tricarboxylic acid (TCA) metabolism and lipogenesis. In contrast to proliferating cells, which use glucose and glutamine for acetyl-coenzyme A (AcCoA) generation, differentiated adipocytes showed increased branched-chain amino acid (BCAA) catabolic flux such that leucine and isoleucine from medium and/or from protein catabolism accounted for as much as 30% of lipogenic AcCoA pools. Medium cobalamin deficiency caused methylmalonic acid accumulation and odd-chain fatty acid synthesis. Vitamin B12 supplementation reduced these metabolites and altered the balance of substrates entering mitochondria. Finally, inhibition of BCAA catabolism compromised adipogenesis. These results quantitatively highlight the contribution of BCAAs to adipocyte metabolism and suggest that BCAA catabolism has a functional role in adipocyte differentiation.

The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales (Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures.

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.

Microbiota of the large intestine synthesizes considerable amount of vitamin B1 (thiamine) in the form of thiamine pyrophosphate (TPP). We have recently demonstrated the existence of an efficient and specific carrier-mediated uptake process for TPP in human colonocytes, identified the TPP transporter (TPPT) involved (product of the SLC44A4 gene), and shown that expression of TPPT along the gastrointestinal (GI) tract is restricted to the colon. Our aim in this study was to determine the molecular basis of the colon-specific expression of TPPT focusing on a possible epigenetic mechanism. Our results showed that the CpG island predicted in the SLC44A4 promoter is non-methylated in the human colonic epithelial NCM460 cells, but is hyper-methylated in the human duodenal epithelial HuTu80 cells (as well as in the human retinal pigment epithelial ARPE19 cells). In the mouse (where TPPT expression in the GI tract is also restricted to the colon), the CpG island predicted in the Slc44a4 promoter is non-methylated in both the jejunum and colon, thus arguing against possible contribution of DNA methylation in the colon-specific expression of TPPT. A role for histone modifications in the tissue-specific pattern of Slc44a4 expression, however, was suggested by the findings that in mouse colon, histone H3 in the 5'-regulatory region of Slc44a4 is tri-methylated at lysine 4 and acetylated at lysine 9, whereas the tri-methylation at lysine 27 modification was negligible. In contrast, in the mouse jejunum, histone H3 is hyper-trimethylated at lysine 27 (repressor mark). Similarly, possible involvement of miRNA(s) in the tissue-specific expression of TPPT was also suggested by the findings that the 3'-UTR of SLC44A4 is targeted by specific miRNAs/RNA binding proteins in non-colonic, but not in colonic, epithelial cells. These studies show, for the first time, epigenetic mechanisms (histone modifications) play a role in determining the tissue-specific pattern of expression of TPPT in the GI tract.

Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.

Synthetic zinc finger proteins (ZFPs) can be created to target promoter DNA sequences, repressing transcription. The binding of small RNA (sRNA) to ZFP mRNA creates an ultrasensitive response to generate higher effective Hill coefficients. Here we combined three "off the shelf" ZFPs and three sRNAs to create new modular inverters in E. coli and quantify their behavior using induction fold. We found a general ordering of the effects of the ZFPs and sRNAs on induction fold that mostly held true when combining these parts. We then attempted to construct a ring oscillator using our new inverters. Our chosen parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.

Previous studies have demonstrated focal but limited molecular similarities between circulating tumor cells (CTCs) and biopsies using isolated genetic assays. We hypothesized that molecular similarity between CTCs and tissue exists at the single cell level when characterized by whole genome sequencing (WGS). By combining the NanoVelcro CTC Chip with laser capture microdissection (LCM), we developed a platform for single-CTC WGS. We performed this procedure on CTCs and tissue samples from a patient with advanced prostate cancer who had serial biopsies over the course of his clinical history. We achieved 30X depth and ≥ 95% coverage. Twenty-nine percent of the somatic single nucleotide variations (SSNVs) identified were founder mutations that were also identified in CTCs. In addition, 86% of the clonal mutations identified in CTCs could be traced back to either the primary or metastatic tumors. In this patient, we identified structural variations (SVs) including an intrachromosomal rearrangement in chr3 and an interchromosomal rearrangement between chr13 and chr15. These rearrangements were shared between tumor tissues and CTCs. At the same time, highly heterogeneous short structural variants were discovered in PTEN, RB1, and BRCA2 in all tumor and CTC samples. Using high-quality WGS on single-CTCs, we identified the shared genomic alterations between CTCs and tumor tissues. This approach yielded insight into the heterogeneity of the mutational landscape of SSNVs and SVs. It may be possible to use this approach to study heterogeneity and characterize the biological evolution of a cancer during the course of its natural history.

High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/.

Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.