Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GapA and GapB. cDNA clones containing the entire coding sequences of the cytosolic precursors for GapA from pea and for GapB from pea and spinach have been identified, sequenced and the derived amino acid sequences have been compared to the corresponding sequences from tobacco, maize and mustard. These comparisons show that GapB differs from GapA in about 20% of its amino acid residues and by the presence of a flexible and negatively charged C-terminal extension, possibly responsible for the observed association of the enzyme with chloroplast envelopes in vitro. This C-terminal extension (29 or 30 residues) may be susceptible to proteolytic cleavage thereby leading to a conversion of chloroplast GAPDH isoenzyme I into isoenzyme II. Evolutionary rate comparisons at the amino acid sequence level show that chloroplast GapA and GapB evolve roughly two-fold slower than their cytosolic counterpart GapC. GapA and GapB transit peptides evolve about 10 times faster than the corresponding mature subunits. They are relatively long (68 and 83 residues for pea GapA and spinach GapB respectively) and share a similar amino acid framework with other chloroplast transit peptides

Aspects of coverage in medical DNA sequencing

<p>Abstract</p> <p>Background</p> <p>DNA sequencing is now emerging as an important component in biomedical studies of diseases like cancer. Short-read, highly parallel sequencing instruments are expected to be used heavily for such projects, but many design specifications have yet to be conclusively established. Perhaps the most fundamental of these is the redundancy required to detect sequence variations, which bears directly upon genomic coverage and the consequent resolving power for discerning somatic mutations.</p> <p>Results</p> <p>We address the medical sequencing coverage problem via an extension of the standard mathematical theory of haploid coverage. The expected diploid multi-fold coverage, as well as its generalization for aneuploidy are derived and these expressions can be readily evaluated for any project. The resulting theory is used as a scaling law to calibrate performance to that of standard BAC sequencing at 8× to 10× redundancy, i.e. for expected coverages that exceed 99% of the unique sequence. A differential strategy is formalized for tumor/normal studies wherein tumor samples are sequenced more deeply than normal ones. In particular, both tumor alleles should be detected at least twice, while both normal alleles are detected at least once. Our theory predicts these requirements can be met for tumor and normal redundancies of approximately 26× and 21×, respectively. We explain why these values do not differ by a factor of 2, as might intuitively be expected. Future technology developments should prompt even deeper sequencing of tumors, but the 21× value for normal samples is essentially a constant.</p> <p>Conclusion</p> <p>Given the assumptions of standard coverage theory, our model gives pragmatic estimates for required redundancy. The differential strategy should be an efficient means of identifying potential somatic mutations for further study.</p

The Cyprinodon variegatus genome reveals gene expression changes underlying differences in skull morphology among closely related species

Genes in durophage intersection set at 15 dpf. This is a comma separated table of the genes in the 15 dpf durophage intersection set. Given are edgeR results for each pairwise comparison. Columns indicating whether a gene is included in the intersection set at a threshold of 1.5 or 2 fold are provided. (CSV 13 kb

Challenges and New Approaches to Proving the Existence of Muscle Synergies of Neural Origin

Muscle coordination studies repeatedly show low-dimensionality of muscle activations for a wide variety of motor tasks. The basis vectors of this low-dimensional subspace, termed muscle synergies, are hypothesized to reflect neurally-established functional muscle groupings that simplify body control. However, the muscle synergy hypothesis has been notoriously difficult to prove or falsify. We use cadaveric experiments and computational models to perform a crucial thought experiment and develop an alternative explanation of how muscle synergies could be observed without the nervous system having controlled muscles in groups. We first show that the biomechanics of the limb constrains musculotendon length changes to a low-dimensional subspace across all possible movement directions. We then show that a modest assumption—that each muscle is independently instructed to resist length change—leads to the result that electromyographic (EMG) synergies will arise without the need to conclude that they are a product of neural coupling among muscles. Finally, we show that there are dimensionality-reducing constraints in the isometric production of force in a variety of directions, but that these constraints are more easily controlled for, suggesting new experimental directions. These counter-examples to current thinking clearly show how experimenters could adequately control for the constraints described here when designing experiments to test for muscle synergies—but, to the best of our knowledge, this has not yet been done

Genotypic resistance testing in HIV by arrayed primer extension

The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5′-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere

Experiences with array-based sequence capture; toward clinical applications

Although sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K Roche NimbleGen, Inc. arrays to zoom in on the protein-coding and immediate intron-flanking sequences of 112 genes, potentially involved in mental retardation and congenital malformation. Captured material was sequenced using Illumina technology. A data analysis pipeline was built that detects sequence variants, positions them in relation to the gene, checks for presence in databases (eg, db single-nucleotide polymorphism (SNP)) and predicts the potential consequences at the level of RNA splicing and protein translation. In the samples analyzed, all known variants were reliably detected, including pathogenic variants from control cases and SNPs derived from array experiments. Although overall coverage varied considerably, it was reproducible per region and facilitated the detection of large deletions and duplications (copy number variations), including a partial deletion in the B3GALTL gene from a patient sample. For ultimate diagnostic application, overall results need to be improved. Future arrays should contain probes from both DNA strands, and to obtain a more even coverage, one could add fewer probes from densely and more probes from sparsely covered regions

Unusual multisystemic involvement and a novel BAG3 mutation revealed by NGS screening in a large cohort of myofibrillar myopathies

Background: Myofibrillar myopathies (MFM) are a group of phenotypically and genetically heterogeneous neuromuscular disorders, which are characterized by protein aggregations in muscle fibres and can be associated with multisystemic involvement. Methods: We screened a large cohort of 38 index patients with MFM for mutations in the nine thus far known causative genes using Sanger and next generation sequencing (NGS). We studied the clinical and histopathological characteristics in 38 index patients and five additional relatives (n = 43) and particularly focused on the associated multisystemic symptoms. Results: We identified 14 heterozygous mutations (diagnostic yield of 37%), among them the novel p.Pro209Gln mutation in the BAG3 gene, which was associated with onset in adulthood, a mild phenotype and an axonal sensorimotor polyneuropathy, in the absence of giant axons at the nerve biopsy. We revealed several novel clinical phenotypes and unusual multisystemic presentations with previously described mutations: hearing impairment with a FLNC mutation, dysphonia with a mutation in DES and the first patient with a FLNC mutation presenting respiratory insufficiency as the initial symptom. Moreover, we described for the first time respiratory insufficiency occurring in a patient with the p.Gly154Ser mutation in CRYAB. Interestingly, we detected a polyneuropathy in 28% of the MFM patients, including a BAG3 and a MYOT case, and hearing impairment in 13%, including one patient with a FLNC mutation and two with mutations in the DES gene. In four index patients with a mutation in one of the MFM genes, typical histological findings were only identified at the ultrastructural level (29%). Conclusions: We conclude that extraskeletal symptoms frequently occur in MFM, particularly cardiac and respiratory involvement, polyneuropathy and/or deafness. BAG3 mutations should be considered even in cases with a mild phenotype or an adult onset. We identified a genetic defect in one of the known genes in less than half of the MFM patients, indicating that more causative genes are still to be found. Next generation sequencing techniques should be helpful in achieving this aim

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease

Increased cholinergic contractions of jejunal smooth muscle caused by a high cholesterol diet are prevented by the 5-HT(4 )agonist – tegaserod

BACKGROUND: Excess cholesterol in bile and in blood is a major risk factor for the respective development of gallbladder disease and atherosclerosis. This lipid in excess negatively impacts the functioning of other smooth muscles, including the intestine. Serotonin is an important mediator of the contractile responses of the small intestine. Drugs targeting the serotonin receptor are used as prokinetic agents to manage intestinal motor disorders, in particular irritable bowel syndrome. Thus, tegaserod, acting on 5-HT(4 )receptor, ideally should obviate detrimental effects of excessive cholesterol on gastrointestinal smooth muscle. In this study we examined the effect of tegaserod on cholesterol-induced changes in the contractile responses of intestinal smooth muscle. METHODS: The effects of a high cholesterol (1%) diet on the in vitro contractile responses of jejunal longitudinal smooth muscle from Richardson ground squirrels to the cholinergic agonist carbachol were examined in the presence or absence of tetrodrodotoxin (TTX). Two groups of animals, fed either low (0.03%) or high cholesterol rat chow diet, were further divided into two subgroups and treated for 28 days with either vehicle or tegaserod. RESULTS: The high cholesterol diet increased, by nearly 2-fold, contractions of the jejunal longitudinal smooth muscle elicited by carbachol. These cholinergic contractions were mediated by muscarinic receptors since they were blocked by scopolamine, a muscarinic receptor antagonist, but not by the nicotinic receptor antagonist, hexamethonium. Tegaserod treatment, which did not affect cholinergic contractions of tissues from low cholesterol fed animals, abrogated the increase caused by the high cholesterol diet. With low cholesterol diet TTX enhanced carbachol-evoked contractions, whereas this action potential blocker did not affect the augmented cholinergic contractions seen with tissues from animals on the high cholesterol diet. Tegaserod-treatment removed the effects of a high cholesterol diet on neuronal muscarinic receptors, as the potentiating effect of TTX on carbachol-elicited contractions was maintained in these animals. CONCLUSION: A high cholesterol diet causes significant changes to cholinergic neurotransmission in the enteric nerves of the jejunum. The mechanisms by which these effects of cholesterol are reversed by tegaserod are unknown, but relate to removal of an inhibitory effect of cholesterol on enteric nerves