Ensembl 2004.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organize biology around the sequences of large genomes. It is a comprehensive and integrated source of annotation of large genome sequences, available via interactive website, web services or flat files. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. The facilities of the system range from sequence analysis to data storage and visualization and installations exist around the world both in companies and at academic sites. With a total of nine genome sequences available from Ensembl and more genomes to follow, recent developments have focused mainly on closer integration between genomes and external data.

Ensembl 2002: accommodating comparative genomics.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of human, mouse and other genome sequences, available as either an interactive web site or as flat files. Ensembl also integrates manually annotated gene structures from external sources where available. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. These range from sequence analysis to data storage and visualisation and installations exist around the world in both companies and at academic sites. With both human and mouse genome sequences available and more vertebrate sequences to follow, many of the recent developments in Ensembl have focusing on developing automatic comparative genome analysis and visualisation.

The Ensembl genome database project.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.

Application of multiple sequence alignment profiles to improve protein secondary structure prediction.

The effect of training a neural network secondary structure prediction algorithm with different types of multiple sequence alignment profiles derived from the same sequences, is shown to provide a range of accuracy from 70.5% to 76.4%. The best accuracy of 76.4% (standard deviation 8.4%), is 3.1% (Q(3)) and 4.4% (SOV2) better than the PHD algorithm run on the same set of 406 sequence non-redundant proteins that were not used to train either method. Residues predicted by the new method with a confidence value of 5 or greater, have an average Q(3) accuracy of 84%, and cover 68% of the residues. Relative solvent accessibility based on a two state model, for 25, 5, and 0% accessibility are predicted at 76.2, 79.8, and 86. 6% accuracy respectively. The source of the improvements obtained from training with different representations of the same alignment data are described in detail. The new Jnet prediction method resulting from this study is available in the Jpred secondary structure prediction server, and as a stand-alone computer program from: http://barton.ebi.ac.uk/. Proteins 2000;40:502-511.

ProtEST: protein multiple sequence alignments from expressed sequence tags.

MOTIVATION: An automatic sequence searching method (ProtEST) is described which constructs multiple protein sequence alignments from protein sequences and translated expressed sequence tags (ESTs). ProtEST is more effective than a simple TBLASTN search of the query against the EST database, as the sequences are automatically clustered, assembled, made non-redundant, checked for sequence errors, translated into protein and then aligned and displayed. RESULTS: A ProtEST search found a non-redundant, translated, error- and length-corrected EST sequence for > 58% of sequences when single sequences from 1407 Pfam-A seed alignments were used as the probe. The average family size of the resulting alignments of translated EST sequences contained > 10 sequences. In a cross-validated test of protein secondary structure prediction, alignments from the new procedure led to an improvement of 3.4% average Q3 prediction accuracy over single sequences. AVAILABILITY: The ProtEST method is available as an Internet World Wide Web service http://barton.ebi.ac.uk/servers/protest.html+ ++ The Wise2 package for protein and genomic comparisons and the ProtESTWise script can be found at http://www.sanger.ac.uk/Software/Wise2 CONTACT: geoff@ebi.ac.uk

Evaluation and improvement of multiple sequence methods for protein secondary structure prediction.

A new dataset of 396 protein domains is developed and used to evaluate the performance of the protein secondary structure prediction algorithms DSC, PHD, NNSSP, and PREDATOR. The maximum theoretical Q3 accuracy for combination of these methods is shown to be 78%. A simple consensus prediction on the 396 domains, with automatically generated multiple sequence alignments gives an average Q3 prediction accuracy of 72.9%. This is a 1% improvement over PHD, which was the best single method evaluated. Segment Overlap Accuracy (SOV) is 75.4% for the consensus method on the 396-protein set. The secondary structure definition method DSSP defines 8 states, but these are reduced by most authors to 3 for prediction. Application of the different published 8- to 3-state reduction methods shows variation of over 3% on apparent prediction accuracy. This suggests that care should be taken to compare methods by the same reduction method. Two new sequence datasets (CB513 and CB251) are derived which are suitable for cross-validation of secondary structure prediction methods without artifacts due to internal homology. A fully automatic World Wide Web service that predicts protein secondary structure by a combination of methods is available via http://barton.ebi.ac.uk/.

JPred: a consensus secondary structure prediction server.

UNLABELLED: An interactive protein secondary structure prediction Internet server is presented. The server allows a single sequence or multiple alignment to be submitted, and returns predictions from six secondary structure prediction algorithms that exploit evolutionary information from multiple sequences. A consensus prediction is also returned which improves the average Q3 accuracy of prediction by 1% to 72.9%. The server simplifies the use of current prediction algorithms and allows conservation patterns important to structure and function to be identified. AVAILABILITY: http://barton.ebi.ac.uk/servers/jpred.h tml CONTACT: geoff@ebi.ac.uk

Relationship between abnormal somite development and axial skeletal defects in rats following heat exposure.

The effects of in vivo heat exposure on gestation day (GD) 10 rat embryos were evaluated on GD 11 to determine the relationships between morphological sequelae following in vivo and in vitro exposures and between effects detected on GD 11 and those observed in postnatal day (PND) 3 pups. Anesthetized rats were exposed to 42 degrees C in a warm air incubator until their rectal temperatures reached 41 degrees C or until a rectal temperature of 42-42.5 degrees C had been maintained for 5 minutes. Heat-exposed embryos exhibited a significant decrease in growth parameters including head length, somite number, and protein content/embryo versus controls. These changes correlated well with in vitro effects from an earlier study (G.L. Kimmel et al., '93). Among the morphological endpoints which were slightly delayed in development were the caudal neural tube, branchial bars, forelimb and hindlimb. The only effect on the embryos that could not be explained as a transient delay in development induced by heat was the induction of unsegmented somites. Additional embryos were exposed to 42 degrees C for 15-20 min in vitro and examined specifically for unsegmented somites, which were observed in 47% of embryos exposed to 42 degrees C in vivo or in vitro. This phenomenon was observed in somites 9-20, i.e., those that give rise to cervical and thoracic vertebrae and ribs. These results correlated well with the axial skeletal malformations observed in PND 3 pups exposed to the same heat treatment (C.A. Kimmel et al., '93).

Skeletal development following heat exposure in the rat.

The effects of gestation day (GD) 10 heat exposure in the rat were studied to determine the temperature-response relationship for the induction of skeletal and other defects. Conscious pregnant rats (Experiment 1) were exposed to various temperatures in a warm air chamber. Body temperature was measured using a rectal probe, and these measurements were confirmed as representing core body temperature in separate animals using telemetric procedures. Those animals whose core body temperature was raised to 41-41.9 degrees C had over 90% malformed pups (examined at postnatal day (PND) 3), and a 25% reduction in the percent of live pups per litter. Animals whose temperature was raised to 39.2-40.9 degrees C had a low incidence of pups with similar types of malformations. The primary types of malformations were of the axial skeleton, consisting of fusions and other abnormalities of the ribs and vertebral elements, and a decrease in the total number of ribs and centra. The acute maternal effects of these temperature increases were signs of heat exhaustion during and 1-2 hr after exposure, but there were no permanent changes in weight gain or other signs. When temperatures were raised to > or = 42 degrees C, all maternal animals died. In a second study (Experiment 2), pregnant rats (from a different supplier) were anesthetized to determine the effect of reducing maternal stress and were exposed to heat as in Experiment 1. Those animals whose core body temperature was raised to 42-42.5 degrees C for 5 min had pups with similar responses to those in Experiment 1 at 41-41.9 degrees C, although the reduction in litter size was not as great. Animals whose temperature was raised to 41 degrees C had a much lower incidence of pups with similar defects, and animals whose temperature was raised to 43 degrees C did not survive. A more detailed analysis of the skeletal defects in Experiment 2 showed rib and vertebral malformations that appear to be related to the stage of somite development at the time of exposure.

Embryonic development in vitro following short-duration exposure to heat.

Gestation day (GD) 10 rat embryos (10-12 somites) were exposed in vitro for 10 to 25 minutes at 42 or 43 degrees C and evaluated 24 hrs later for alterations in growth and specific morphological parameters, using a modified Brown-Fabro (Brown and Fabro: Teratology, 24:65-78, '81) scoring system that allowed evaluation of development relative to gestational age. At 42 degrees C, crown-rump length appeared to be particularly sensitive, responding to only 10 mins exposure. A 15-min exposure resulted in decreased total protein, somite number and morphological score. No system was uniquely sensitive, since all parameters demonstrated some degree of response. Rather, systems affected were those that would be developing most rapidly at this time in gestation. At 43 degrees C, all of the parameters measured were affected by a 10-min exposure. These results demonstrate alterations in vitro after much shorter exposure periods than previously reported on GD10, which may be due, in part, to the use of a modified scoring system that permitted the evaluation of graded individual end point changes relative to gestational age. The response patterns demonstrated a clear temperature- and exposure duration-dependency, with a shift from a more shallow duration-response curve to a more dramatic inhibition of development as temperature increased from 42 degrees C to 43 degrees C.

The early effects of ouabain on potassium metabolism and rate of proliferation of mouse lymphoblasts.

Murine lymphoblasts grown in suspension culture in the presence of ouabain showed a dose dependent and sequential decrease in 86Rb+ (K+ analogue) influx, cellular potassium content, and growth rate. An increase in eosin staining and a decrease in cell number was observed after two hours in the presence of 1 mM ouabain; 1 muM ouabain was without effect on any of the parameters measured. Ouabain inhibition was rapidly and completely reversible at concentrations that were not cytotoxic.

Adaptation of potassium metabolism and restoration of mitosis during prolonged treatment of mouse lymphoblasts with ouabain.

The effects of ouabain on the growth of murine lymphoblasts in vitro have been studied. Exposure of cells to ouabain (0.1 mM) initially inhibited 86Rb+ uptake rate, reduced the intracellular potassium concentration, and decreased population growth rates. Continued exposure to the same ouabain concentration resulted in an increase of 86Rb+ uptake rate, intracellular potassium content and population growth rates to control values (adaptation). When treated cells were resuspended in medium free of ouabain after 12 to 15 hours of ouabain treatment, 86Rb+ uptake rates and intracellular potassium levels exceeded those of untreated cells. Adaptation was inhibited by cycloheximide (3 mug/ml) and by actinomycin D (0.05 mug/ml). Kinetic analysis of transport suggested that while the total capacity of the Na/, K+ transport system increased, the affinity for both the cation (86Rb+) and ouabain decreased.

The effects of ouabain on the cell mitotic cycle of mouse lymphoblasts.

The inhibition of cell proliferation by ouabain has been analyzed with respect to the cell cycle. Three lines of evidence indicate that growth rate is modified by altering to different degrees the rate of progress through stages of the cell cycle: (1) a three hour lag occurs between the time of ouabain addition and the inhibition of proliferation; (2) oubain must be present at least two to four hours prior to the mitotic burst of synchronized cells for inhibition of mitosis to occur; (3) parasynchrony is observed when cells are resuspended in ouabain-free medium after 12 hours of exposure to ouabain. Analysis of the distribution of cells in each of the stages of the cell cycle at various times during ouabain treatment reveals a progressive increase in the fraction of cells in S with a concomitant decrease in the percent of cells in each of the stages. These results indicate that the prolongation of the cell cycle time in the presence of ouabain is due primarily to an S stage block.