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.

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.