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.

Apollo: a sequence annotation editor.

The well-established inaccuracy of purely computational methods for annotating genome sequences necessitates an interactive tool to allow biological experts to refine these approximations by viewing and independently evaluating the data supporting each annotation. Apollo was developed to meet this need, enabling curators to inspect genome annotations closely and edit them. FlyBase biologists successfully used Apollo to annotate the Drosophila melanogaster genome and it is increasingly being used as a starting point for the development of customized annotation editing tools for other genome projects.

A comparison of two murine monoclonal antibodies humanized by CDR-grafting and variable domain resurfacing.

The variable domain resurfacing and CDR-grafting approaches to antibody humanization were compared directly on the two murine monoclonal antibodies N901 (anti-CD56) and anti-B4 (anti-CD19). Resurfacing replaces the set of surface residues of a rodent variable region with a human set of surface residues. The method of CDR-grafting conceptually consists of transferring the CDRs from a rodent antibody onto the Fv framework of a human antibody. Computer-aided molecular modeling was used to design the initial CDR-grafted and resurfaced versions of these two antibodies. The initial versions of resurfaced N901 and resurfaced anti-B4 maintained the full binding affinity of the original murine parent antibodies and further refinements to these versions described herein generated five new resurfaced antibodies that contain fewer murine residues at surface positions, four of which also have the full parental binding affinity. A mutational study of three surface positions within 5 A of the CDRs of resurfaced anti-B4 revealed a remarkable ability of the resurfaced antibodies to maintain binding affinity despite dramatic changes of charges near their antigen recognition surfaces, suggesting that the resurfacing approach can be used with a high degree of confidence to design humanized antibodies that maintain the full parental binding affinity. By comparison CDR-grafted anti-B4 antibodies with parental affinity were produced only after seventeen versions were attempted using two different strategies for selecting the human acceptor frameworks. For both the CDR-grafted anti-B4 and N901 antibodies, full restoration of antigen binding affinity was achieved when the most identical human acceptor frameworks were selected. The CDR-grafted anti-B4 antibodies that maintained high affinity binding for CD19 had more murine residues at surface positions than any of the three versions of the resurfaced anti-B4 antibody. This observation suggests that the resurfacing approach can be used to produce humanized antibodies with reduced antigenic potential relative to their corresponding CDR-grafted versions.

Inorganic pyrophosphatase of Trichomonas vaginalis.

Trichomonas vaginalis homogenates were found to have an acid inorganic pyrophosphatase activity with a specific activity at pH 4.8 of about 7 nmol min-1 (mg protein)-1. This activity was localized predominantly in hydrolase containing particles, showed structure-bound latency and was tightly membrane-bound. The activity showed no magnesium dependence, a Km of about 2 mM inorganic pyrophosphate, a pH optimum of 5.2 and was inhibited by fluoride at millimolar levels. No evidence was obtained for the existence of a cytosolic magnesium-dependent activity but the existence of a low level of magnesium-independent cytosolic activity cannot be excluded. These observations correlate with the importance of cytosolic inorganic pyrophosphate in the carbohydrate catabolism in T. vaginalis.