The Vertebrate Genome Annotation (Vega) database.

The Vertebrate Genome Annotation (Vega) database (http://vega.sanger.ac.uk) has been designed to be a community resource for browsing manual annotation of finished sequences from a variety of vertebrate genomes. Its core database is based on an Ensembl-style schema, extended to incorporate curation-specific metadata. In collaboration with the genome sequencing centres, Vega attempts to present consistent high-quality annotation of the published human chromosome sequences. In addition, it is also possible to view various finished regions from other vertebrates, including mouse and zebrafish. Vega displays only manually annotated gene structures built using transcriptional evidence, which can be examined in the browser. Attempts have been made to standardize the annotation procedure across each vertebrate genome, which should aid comparative analysis of orthologues across the different finished regions.

The DNA sequence and comparative analysis of human chromosome 10.

The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.

The DNA sequence and analysis of human chromosome 13.

Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.

The DNA sequence and analysis of human chromosome 6.

Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.

Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.

The DNA sequence and comparative analysis of human chromosome 20.

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

Stuttering: neuropsychiatric features measured by content analysis of speech and the effect of risperidone on stuttering severity.

Positron-emission tomographic (PET) studies and genetic research of stuttering have recently revealed underlying cerebral neurobiologic contributing factors in this disorder. We aimed to assess whether cognitive impairment and other neuropsychiatric dimensions could be detected through computerized content analysis of short samples of speech from stutterers, and whether administration of risperidone in a double-blind placebo-controlled study could decrease the severity of stuttering, as well as any of the neuropsychiatric features of these stutterers. A group of 21 stutterers with the developmental form of stuttering, an onset before age of 6 years, aged 20 to 74 years, and who were otherwise free of major medical or psychiatric problems, initially gave a 5-minute tape-recorded speech sample in response to purposely ambiguous instructions to talk about any interesting or dramatic life experiences. Then, half of these subjects (n = 10) were randomly selected to receive 6 weeks of risperidone treatment up to 2.0 mg/d and the other half (n = 11) were administered a placebo. Both groups of subjects gave a second verbal sample after 6 weeks of treatment. Significantly elevated cognitive impairment and social alienation-personal disorganization scores, derived from the computerized content of the initial 5-minute speech samples, were found. After 6 weeks, the risperidone group improved significantly on a measure of severity of stuttering but did not improve on the percentage of time spent stuttering. The placebo group did not improve on either measure of stuttering. The psychopathological processes of subjects who received risperidone treatment, including those with elevated cognitive impairment and social alienation-personal disorganization, did not change significantly. However, stutterers who had lower scores on verbal content analysis-derived shame anxiety, guilt anxiety, or hostility inward measures improved significantly more with risperidone than stutterers with higher scores on these measures. The findings of elevated cognitive impairment and social alienation-personal disorganization scores of adult stutterers with the early developmental form of stuttering are consistent with the neurobiologic abnormalities found in PET-scan and genetic research involving stutterers. Risperidone (< or =2.0 mg/d) can reduce the severity of stuttering while not significantly affecting the magnitude of neuropsychiatric dimensions such as cognitive impairment or social alienation-personal disorganization. The less the inward shame, guilt, or hostility of the stutterers, the better the beneficial effect of risperidone on the severity of stuttering.

Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels.

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.

Characterization of pheophytin ground states in Rhodobacter sphaeroides R26 photosynthetic reaction centers from multispin pheophytin enrichment and 2-D 13C MAS NMR dipolar correlation spectroscopy.

The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from 13C chemical shift data. A new experimental approach involving multispin 13C labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly 13C biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state 13C magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state 13C-13C correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many 13C resonances of the [U-13C]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of 13C shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.

Intraventricular hemorrhage and high-frequency ventilation: a meta-analysis of prospective clinical trials.

OBJECTIVE: The association between high-frequency ventilation (HFV) and intraventricular hemorrhage (IVH) and periventricular leukomalacia (PVL) has been debated. PURPOSE: To determine if premature neonates treated with HFV are at greater risk for developing IVH and/or PVL than neonates treated with conventional ventilation, we completed a meta-analysis of all prospective randomized control trials comparing HFV and conventional ventilation in the management of respiratory distress syndrome. METHODS: The meta-analysis included nine studies comparing HFV and conventional ventilation in the management of preterm neonates. To summarize the data, we calculated the difference in absolute risk for IVH and PVL between neonates treated with HFV and those treated with standard ventilation. These differences were combined to determine an overall difference in the absolute risk and its confidence interval. We examined the effect of estimated gestational age, birth weight, surfactant, and age at study entry on the results. Because one trial (HIFI study) was much larger than the other studies, it dominated the analysis, so we evaluated the data with and without including data from the HIFI trial. RESULTS: The occurrences of IVH and PVL ranged from 14% to 47% and 5% to 16%, respectively. This variation may be explained by the difference in the populations of neonates treated. The meta-analysis showed that use of HFV was associated with an increased risk of PVL (odds ratio = 1.7 with a confidence interval of 1.06 to 2.74), but not IVH or severe (> or = grade 3) IVH. When the results of the HIFI study were excluded, there were no differences between HFV and conventional ventilation in the occurrence of IVH or PVL. CONCLUSIONS: The association between HFV and adverse neurologic outcomes is primarily influenced by the results of the HIFI trial. Meta-analysis of more recent studies does not confirm the findings of the HIFI trial and suggests that HFV is not associated with increased occurrence of IVH or PVL.

Plant viral leaders influence expression of a reporter gene in tobacco.

In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter beta-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two- to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four- to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four- to six-fold more efficiently than the random leader control.

Night-to-night variability in sleep apnea and sleep-related periodic leg movements in the elderly.

The amount of night-to-night variability in sleep apnea (SA) and sleep-related periodic leg movements (PLMs) is largely unknown but, despite this, clinical decisions are based on single-night studies in many clinical sleep laboratories. We examined variability in SA and PLMs over three nights in 46 community-resident seniors. No evidence was found for either a first-night effect or a directional trend across nights in either the Respiratory Disturbance Index (RDI) or the Movement Index (MI), despite a prominent first-night effect on pattern of sleep. Duration of apneas/hypopneas and degree of associated heart rate change and oxygen desaturation in subjects with SA and intermovement interval in subjects with PLMs also failed to show systematic change across nights. However, if a cut-off score of 5/h for RDI and MI was used, the classification recorded on the first night did differ from the classification given on at least one of the other nights in 43% of the subjects. The magnitude of fluctuation in RDI or MI from night to night was large enough in some subjects that, in a clinical situation, decisions based on one night would have been entirely different had the subject been studied on a different night. Night-to-night variability in RDI and MI within subjects also was associated with significant alterations in the sleep pattern. We conclude that caution should be taken in drawing conclusions from single-night studies, especially in individuals with relatively mild forms of SA and PLMs where nightly variations could easily place them above or below an arbitrary cut-off score.

Computer-derived equations for predicting survival postoperatively. Their usefulness and limitations.

We used multivariate analysis to determine whether survival following perforations of the gastrointestinal tract could be accurately predicted from preoperative data. Of 12 variables tested, four were found to have predictive value. These were age, pulmonary disease, preoperative shock, and the attending surgeon. When these four variables were employed in a logistic regression equation on 42 patients, it correctly predicted which 21 patients died before leaving the hospital. To produce an equation useful for other hospitals, we recalculated it without the attending surgeon variable. Again, the equation was used to predict survival. The correlation of predicted vs observed outcome remained high, and, using a 2 x 2 chi 2 test, the correlation was significant. We then cross validated the three-variable model on data from a second hospital. The model accurately predicted the new data equally well. We believe that predictive models can identify risk factors in a variety of patient populations and can determine who is likely to benefit from specific treatment modalities.