Splign: algorithms for computing spliced alignments with identification of paralogs

Background  

The computation of accurate alignments of cDNA sequences against a genome is at the foundation of modern genome annotation pipelines. Several factors such as presence of paralogs, small exons, non-consensus splice signals, sequencing errors and polymorphic sites pose recognized difficulties to existing spliced alignment algorithms.     

Inhibition of a Glucose-Limited Sequencing Fed-Batch Culture of Salmonella enterica Serovar Enteritidis by Volatile Fatty Acids Representative of the Ceca of Broiler Chickens

The effects of concentrations of volatile fatty acids on an anaerobic, glucose-limited, and pH-controlled growing culture of Salmonella enterica serovar Enteritidis were studied. Suddenly increasing volatile fatty acids to the concentrations representative of the ceca of 15-day-old broiler chickens caused washout of serovar Enteritidis. In contrast, a sudden increase to the volatile fatty acid concentrations representative of the ceca of younger broiler chickens caused a reduction in the biomass but not washout. Gradually increasing volatile fatty acids caused a gradual decrease in the biomass of serovar Enteritidis. We conclude that the concentrations of volatile fatty acids present in the ceca of broilers with a mature microflora can cause washout of serovar Enteritidis in an in vitro system mimicking cecal ecophysiology.     

Familial megacecum and colon in the rat: a new model of gastrointestinal neuromuscular dysfunction

Gastrointestinal motility disorders are of considerable clinical importance in humans and animals. Abnormalities of smooth muscle and the enteric nervous system have been described. We have identified and characterized a new mutant stock of rats that develops severe megacecum and colon with pseudo-obstruction, Familial Megacecum and Colon (FMC). The inheritance pattern of FMC was characterized by selective breeding. Gastrointestinal motility was evaluated radiographically. Complete pathologic evaluations, including ultrastructural examination and staining of colonic segments for acetylcholinesterase, peripherin, vasoactive intestinal peptide, substance P, nitric oxide synthase, and somatostatin, were performed. Spontaneous contractility and contractile force in isolated colonic muscle strips were examined. Familial megacecum and colon is inherited as an autosomal recessive trait. The markedly dilated cecum and proximal portion of the colon are followed by a short, funnel-shaped segment and distal portion of the colon with normal or slightly reduced lumen. Although clinical features and gross anatomic changes of the colon resemble those of Hirschsprung's disease in humans and animals, aganglionosis is not a feature of FMC. An increase in somatostatin staining was observed in dilated regions of bowel. The spontaneous contractile frequency and contractile force were diminished in the affected colon. Familial megacecum and colon is a new mutant, distinct from previously described hereditary and targeted mutant rodent models that develop megacecum and colon as a result of distal colonic dysfunction. The functional or morphologic defect(s) that result in colonic dysfunction in rats with FMC was not determined. The disease may result from an absence or overexpression of a single or group of neurotransmitters or their respective neurons, receptor abnormalities, or defects in the intestinal pacemaker system.     

NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A).d(T-A-C-G-T-A) duplex

Two-dimensional homonuclear and heteronuclear NMR and minimized potential energy calculations have been combined to define the structure of the antitumor agent mitomycin C (MC) cross-linked to deoxyguanosines on adjacent base pairs in the d(T1-A2-C3-G4-T5-A6).d(T7-A8-C9-G10-T11-A12) duplex. The majority of the mitomycin and nucleic acid protons in the MC-X 6-mer complex have been assigned from through-bond and through-space two-dimensional proton NMR studies in aqueous solution at 5 and 20 degrees C. The C3.G10 and G4.C9 base pairs are intact at the cross-link site and stack on each other in the complex. The amino protons of G4 and G10 resonate at 9.36 and 8.87 ppm and exhibit slow exchange with solvent H2O. The NMR experimental data establish that the mitomycin is cross-linked to the DNA through the amino groups of G4 and G10 and is positioned in the minor groove. The conformation of the cross-link site is defined by a set of NOEs between the mitomycin H1" and H2" protons and the nucleic acid imino and amino protons of G4 and the H2 proton of A8 and another set of NOEs between the mitomycin geminal H10" protons and the nucleic acid imino and amino protons of G10 and the H2 proton of A2. Several phosphorus resonances of the d(T-A-C-G-T-A) duplex shift dramatically on mitomycin cross-link formation and have been assigned from proton-detected phosphorus-proton two-dimensional correlation experiments. The proton chemical shifts and NOEs establish fraying at the ends of the d(T-A-C-G-T-A) duplex, and this feature is retained on mitomycin cross-link formation. The base-base and base-sugar NOEs exhibit similar patterns for symmetry-related steps on the two nucleic acid strands in the MC-X 6-mer complex, while the proton and phosphorus chemical shifts are dramatically perturbed at the G10-T11 step on cross-link formation. The NMR distance constraints have been included in minimized potential energy computations on the MC-X 6-mer complex. These computations were undertaken with the nonplanar five-membered ring of mitomycin in each of two pucker orientations. The resulting low-energy structures MX1 and MX2 have the mitomycin cross-linked in a widened minor groove with the chromophore ring system in the vicinity of the G10-T11 step on one of the two strands in the duplex.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structure of postsynaptic densities isolated from dog cerebral cortex: I. overall morphology and protein composition

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.     

The structure of postsynaptic densities isolated from dog cerebral cortex: II. characterization and arrangement of some of the major proteins within the structure

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ extractor, ethylene glycol-bis (beta- aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.