The distribution of intra-genomically variable dinoflagellate symbionts at Lord Howe Island,Australia

The symbiotic dinoflagellates of corals and other marine invertebrates (Symbiodinium) are essential to the development of shallow-water coral reefs. This genus contains considerable genetic diversity and a corresponding range of physiological and ecological traits. Most genetic variation arises through the accumulation of somatic mutations that arise during asexual reproduction. Yet growing evidence suggests that occasional sexual reproductive events also occur within, and perhaps between, Symbiodinium lineages, further contributing to the pool of genetic variation available for evolutionary adaptation. Intra-genomic variation can therefore arise from both sexual and asexual reproductive processes, making it difficult to discern its underlying causes and consequences. We used quantitative PCR targeting the ITS2 locus to estimate proportions of genetically homogeneous symbionts and intra-genomically variable Symbiodinium (IGV Symbiodinium) in the reef-building coral Pocillopora damicornis at Lord Howe Island, Australia. We then sampled colonies through time and at a variety of spatial scales to find out whether the distribution of these symbionts followed patterns consistent with niche partitioning. Estimated ratios of homogeneous to IGV Symbiodinium varied between colonies within sites (metres to tens of metres) and between sites separated by hundreds to thousands of metres, but remained stable within colonies through time. Symbiont ratios followed a temperature gradient, with the local thermal maximum emerging as a negative predictor for the estimated proportional abundance of IGV Symbiodinium. While this pattern may result from fine-scale spatial population structure, it is consistent with an increased susceptibility to thermal stress, suggesting that the evolutionary processes that generate IGV (such as inter-lineage recombination and the accumulation of somatic mutations at the ITS2 locus) may have important implications for the fitness of the symbiont and that of the coral host.     

Together stronger: Intracolonial genetic variability occurrence in Pocillopora corals suggests potential benefits

We investigated the occurrence of intracolonial genetic variability (IGV) in Pocillopora corals in the southwestern Indian Ocean. Ninety‐six colonies were threefold‐sampled from three sites in Reunion Island. Nubbins were genotyped using 13 microsatellite loci, and their multilocus genotypes compared. Over 50% of the colonies presented at least two different genotypes among their three nubbins, and IGV was found abundant in all sites (from 36.7% to 58.1%). To define the threshold distinguishing mosaicism from chimerism, we developed a new method based on different evolution models by computing the number of different alleles for the infinite allele model (IAM) and the Bruvo's distance for the stepwise mutation model (SMM). Colonies were considered as chimeras if their nubbins differed from more than four alleles and if the pairwise Bruvo's distance was higher than 0.12. Thus 80% of the IGV colonies were mosaics and 20% chimeras (representing almost 10% of the total sampling). IGV seems widespread in scleractinians and beyond the disabilities of this phenomenon reported in several studies, it should also bring benefits. Next steps are to identify these benefits and to understand processes leading to IGV, as well as factors influencing them.     

Precise Identification of the Introgressed Segments in Powdery Mildew-resistant Introgression Lines of Triticum aestivum-T.timopheevi

On the basis of the reported location of the Pm6 gene, 36 homoeologous group-2 specific probes were selected to detect polymorphism between wheat/Triticum timopheevi Zhuk. Pm6 introgression lines and their recurrent parent "Prins". Two Pm6 introgression lines IGV1-456, IGV1-458 were distinguished from the others. Nineteen long ann specific and six short ann specific probes detected the RFLPs between resistant IGV1456, IGV1-458 and susceptible control "Prins", indicated that the introgressed T. timopheevi 2G chromatin involve beth arms spanning across the centromere between markers Xcdo405 and Xbcd135. Only 6 of the nineteen long ann specific probes and two additional probes－BCD292, CDO678 showed RFLPs between chromosome 2B of "Prins" and IGV1-463. This means that the introgressed T. timopheevi segment in IGV1- 463 with breakpoints between markers Xbcd307 and Xcdo678 is smaller than those detected in IGV1-456 and IGV1-458. Two of the six long arm specific probes PSR934, BCD135 detected polymorphism between IGV1- 464 and "Pr ins", and only one clone BCD135 revealed RFLPs between IGV1-465 and "Pr ins", which indicated that the introgressed segments in these two lines are smaller than those in others. As the introgressed segments in all the introgression lines bear the Pm6 gene, after comparison of the overlaps of the introgressed segments, it might be reasonable to map the gene Pm6 in the region of marker Xbcd135-2BL flanked 2BL.     

利用基于PCR的分子标记区分普通小麦-提莫菲维小麦渐渗系

采用定位于小麦2B染色体上的72对分子标记对含小麦抗白粉病基因Pm6的8份普通小麦（T.aestivum L.）-提莫菲维（T.timopheevii zhuk.）渐渗系材料进行分析, 通过分子标记标图确定8份材料中渗入的提莫菲维小麦染色体片段的大小, 同时结合连锁图谱对这些材料进行了遗传和物理标图。参考本研究所用的分子标记在染色体2B上的定位结果, Pm6基因被位于2B 染色体长臂近末端2BL-6区域, 提莫菲维小麦2G染色体渐渗片断长度由短到长排列顺序为: IGV1-465相似文献   

Genetic mapping of the powdery mildew resistance gene Pm6 in wheat by RFLP analysis

Pm6 in bread wheat (Triticum aestivum L.), which was transferred from Triticum. timopheevii L., is a gene conferring resistance to the powdery mildew disease caused by Erysiphe graminis f. sp. tritici. Six near-isogenic lines ( NILs ) of Pm6 in a cultivar ’Prins’ background were analyzed to map this gene using restriction fragment length polymorphism (RFLP). Each of the six NILs possessed a T. timopheevii-derived segment, varying in length, and associated with powdery mildew resistance. Lines IGV1–465 (FAO163b/ 7*Prins) and IGV1–467 (Idaed 59B/7*Prins) had the shortest introgressed segments, which were detected only by DNA probes BCD135 and PSR934, respectively. The polymorphic loci detected by both probes were mapped to the long arm of chromosome 2B. Lines IGV1–458 (CI13250/7*Prins) and IGV1–456 (CI12559/8*Prins) contained the longest T. timopheevii segments involving both arms of donor chromosome 2G across the centromere. All these introgressed segments had an overlapping region flanked by the loci xpsr934 and xbcd135 on 2BL. Thus, Pm6 was located in this region since the powdery mildew resistance in all the NILs resulted from the introgressed fragments. Using the F₂ mapping population from a cross of IGV1–463 (PI170914/7*Prins)×Prins, Pm6 was shown to be closely linked to the loci xbcd135 and xbcd266 at a genetic distance of 1.6 cM and 4.8 cM, respectively. BCD135 was successfully used in detecting the presence of Pm6 in different genetic backgrounds. Received: 29 June 1999 / Accepted: 6 July 1999     

Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em Thell.) 6. Alleles at the Pm5 locus

Genetic characterization of powdery mildew resistance genes were conducted in common wheat cultivars Hope and Selpek possessing resistance gene Pm5, cvs. Ibis and Kormoran expressing resistance gene Mli, a backcross-derived line IGV 1–455 and a Triticum sphaerococcum var. rotundatum Perc. line Kolandi. Monosomic analyses revealed that one major recessive gene is located on chromosome 7B in the lines IGV 1–455 and Kolandi. Allelism tests of the F₂ and F₃ populations involving the tested resistant lines crossed with either cv. Hope or Selpek indicated that their resistance genes are alleles at the Pm5 locus. The alleles are now designated Pm5a in Hope and Selpek, Pm5b in Ibis and Kormoran, Pm5c in T. sphaerococcum var. rotundatum line Kolandi, and Pm5d in backcross-derived line IGV 1–455, respectively. Received: 5 November 1999 / Accepted: 14 April 2000     

Lineage tree analysis of immunoglobulin variable-region gene mutations in autoimmune diseases: chronic activation, normal selection

Autoimmune diseases show high diversity in the affected organs, clinical manifestations and disease dynamics. Yet they all share common features, such as the ectopic germinal centers found in many affected tissues. Lineage trees depict the diversification, via somatic hypermutation (SHM), of immunoglobulin variable-region (IGV) genes. We previously developed an algorithm for quantifying the graphical properties of IGV gene lineage trees, allowing evaluation of the dynamical interplay between SHM and antigen-driven selection in different lymphoid tissues, species, and disease situations. Here, we apply this method to ectopic GC B cell clones from patients with Myasthenia Gravis, Rheumatoid Arthritis, and Sj?gren's Syndrome, using data scaling to minimize the effects of the large variability due to methodological differences between groups. Autoimmune trees were found to be significantly larger relative to normal controls. In contrast, comparison of the measurements for tree branching indicated that similar selection pressure operates on autoimmune and normal control clones.     

Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.     

From diversity to delivery: the case of the Indian Genome Variation initiative

India currently has the world's second-largest population along with a fast-growing economy and significant economic disparity. It also continues to experience a high rate of infectious disease and increasingly higher rates of chronic diseases. However, India cannot afford to import expensive technologies and therapeutics nor can it, as an emerging economy, emulate the health-delivery systems of the developed world. Instead, to address these challenges it is looking to biotechnology-based innovation in the field of genomics. The Indian Genome Variation (IGV) consortium, a government-funded collaborative network among seven local institutions, is a reflection of these efforts. The IGV has recently developed the first large-scale database of genomic diversity in the Indian population that will facilitate research on disease predisposition, adverse drug reactions and population migration.     

Non-linear fitting program for biological data

A simple microcomputer program written in Microsoft Basic estimates pharmacokinetic parameters using the coordinate search technique to minimize the sum of squared errors. The program developed for portable computers combines a plot of data and curve fitting so as to find rapidly the initial parameters with the subsequent optimization of the parameter estimate.     

De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V_L and three V_H domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V_H by keeping V_L in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V_H diversity was sufficient for isolating Fabs to most antigens, whereas variability in V_L was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V_L diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.     

Application of Massively Parallel Sequencing to Genetic Diagnosis in Multiplex Families with Idiopathic Sensorineural Hearing Impairment

Despite the clinical utility of genetic diagnosis to address idiopathic sensorineural hearing impairment (SNHI), the current strategy for screening mutations via Sanger sequencing suffers from the limitation that only a limited number of DNA fragments associated with common deafness mutations can be genotyped. Consequently, a definitive genetic diagnosis cannot be achieved in many families with discernible family history. To investigate the diagnostic utility of massively parallel sequencing (MPS), we applied the MPS technique to 12 multiplex families with idiopathic SNHI in which common deafness mutations had previously been ruled out. NimbleGen sequence capture array was designed to target all protein coding sequences (CDSs) and 100 bp of the flanking sequence of 80 common deafness genes. We performed MPS on the Illumina HiSeq2000, and applied BWA, SAMtools, Picard, GATK, Variant Tools, ANNOVAR, and IGV for bioinformatics analyses. Initial data filtering with allele frequencies (<5% in the 1000 Genomes Project and 5400 NHLBI exomes) and PolyPhen2/SIFT scores (>0.95) prioritized 5 indels (insertions/deletions) and 36 missense variants in the 12 multiplex families. After further validation by Sanger sequencing, segregation pattern, and evolutionary conservation of amino acid residues, we identified 4 variants in 4 different genes, which might lead to SNHI in 4 families compatible with autosomal dominant inheritance. These included GJB2 p.R75Q, MYO7A p.T381M, KCNQ4 p.S680F, and MYH9 p.E1256K. Among them, KCNQ4 p.S680F and MYH9 p.E1256K were novel. In conclusion, MPS allows genetic diagnosis in multiplex families with idiopathic SNHI by detecting mutations in relatively uncommon deafness genes.     

A convenient program for the rapid calculation of sedimentation coefficients in linear salt or sucrose gradients

A program is presented for calculating s_20,w values from data obtained by zone sedimentation in linear sucrose or salt gradients in a variety of rotors at temperatures ranging from 0° to 20°C. The program can be either run with the use of high-speed computers or performed with the aid of a small calculator in a reasonably short time.     

A program for the numerical integration of enzyme kinetic equations using small computers

A simple program to integrate differential equations is given. The program is written in BASIC and has been devised to be used on small computers. The program has been used to integrate the differential equations corresponding to a single reaction-single enzyme, a bireaction-single enzyme and a four reaction-four substrate-single enzyme mechanism: it has been used also for studying the interconversions among various molecular forms of a single enzyme, viz. monomer - dimer - trimer - tetramer. The program has proven to be very effective in these models and its time consumption comparable with that of other longer programs. The BASIC version is shown, and the FORTRAN VS version is available upon request to the authors.     

A novel approach for large-scale polypeptide folding based on elastic networks using continuous optimization

We present a new computationally efficient method for large-scale polypeptide folding using coarse-grained elastic networks and gradient-based continuous optimization techniques. The folding is governed by minimization of energy based on Miyazawa-Jernigan contact potentials. Using this method we are able to substantially reduce the computation time on ordinary desktop computers for simulation of polypeptide folding starting from a fully unfolded state. We compare our results with available native state structures from Protein Data Bank (PDB) for a few de-novo proteins and two natural proteins, Ubiquitin and Lysozyme. Based on our simulations we are able to draw the energy landscape for a small de-novo protein, Chignolin. We also use two well known protein structure prediction software, MODELLER and GROMACS to compare our results. In the end, we show how a modification of normal elastic network model can lead to higher accuracy and lower time required for simulation.     

Simulation of neurocomputing based on the photophobic reactions of Euglena with optical feedback stimulation

To explore possible forms of unconventional computers that have high capacities for adaptation and exploration, we propose a new approach to developing a biocomputer based on the photophobic reactions of microbes (Euglena gracilis), and perform the Monte-Carlo simulation of Euglena-based neural network computing, involving virtual optical feedback to the Euglena cells. The photophobic reactions of Euglena are obtained experimentally, and incorporated in the simulation, together with a feedback algorithm with a modified Hopfield–Tank model for solving a 4-city traveling salesman problem. The simulation shows high performances in terms of (1) reaching one of the best solutions of the problem, and (2) searching for a number of solutions via dynamic transition among the solutions. This dynamic transition is attributed to the fluctuation of state variables, global oscillation through feedback instability, and the one-by-one change of state variables.     

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes,providing for a powerful therapeutic antibody platform

Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1β and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.     

Nucleic acids for the rational design of reaction circuits

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Highlights► Nucleic acids can be used to build reaction modules modeled after in vivo or in silico computers. ► In vitro circuits built from these modules perform one-shot calculations or show complex dynamics. ► Applications at the interface with biology, or for molecular-scale robotics, are burgeoning.