SAND, a new protein family: from nucleic acid to protein structure and function prediction.

As a result of genome, EST and cDNA sequencing projects, there are huge numbers of predicted and/or partially characterised protein sequences compared with a relatively small number of proteins with experimentally determined function and structure. Thus, there is a considerable attention focused on the accurate prediction of gene function and structure from sequence by using bioinformatics. In the course of our analysis of genomic sequence from Fugu rubripes, we identified a novel gene, SAND, with significant sequence identity to hypothetical proteins predicted in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, a Drosophila melanogaster gene, and mouse and human cDNAs. Here we identify a further SAND homologue in human and Arabidopsis thaliana by use of standard computational tools. We describe the genomic organisation of SAND in these evolutionarily divergent species and identify sequence homologues from EST database searches confirming the expression of SAND in over 20 different eukaryotes. We confirm the expression of two different SAND paralogues in mammals and determine expression of one SAND in other vertebrates and eukaryotes. Furthermore, we predict structural properties of SAND, and characterise conserved sequence motifs in this protein family.

Identification of the C3b binding site in a recombinant vWF-A domain of complement factor B by surface-enhanced laser desorption-ionisation affinity mass spectrometry and homology modelling: implications for the activity of factor B.

Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface-enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH3) in the presence of Mg(2+). A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH3), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH3) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH3). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central beta-sheet of the factor B vWF-A structure. The peptides G229-K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A beta-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions.

Generation and analysis of 25 Mb of genomic DNA from the pufferfish Fugu rubripes by sequence scanning.

We have generated and analyzed >50,000 shotgun clones from 1059 Fugu cosmid clones. All sequences have been minimally edited and searched against protein and DNA databases. These data are all displayed on a searchable, publicly available web site at. With an average of 50 reads per cosmid, this is virtually nonredundant sequence skimming, covering 30%-50% of each clone. This essentially random data set covers nearly 25 Mb (>6%) of the Fugu genome and forms the basis of a series of whole genome analyses which address questions regarding gene density and distribution in the Fugu genome and the similarity between Fugu and mammalian genes. The Fugu genome, with eight times less DNA but a similar gene repertoire, is ideally suited to this type of study because most cosmids contain more than one identifiable gene. General features of the genome are also discussed. We have made some estimation of the syntenic relationship between mammals and Fugu and looked at the efficacy of ORF prediction from short, unedited Fugu genomic sequences. Comparative DNA sequence analyses are an essential tool in the functional interpretation of complex vertebrate genomes. This project highlights the utility of using the Fugu genome in this kind of study.

Sequence scanning chicken cosmids: a methodology for genome screening.

The chicken genome is relatively poorly studied at the molecular level. The karyotype 2n=78 is divided into three main chromosomal sub-groups: the macrochromosomes (six pairs), the intermediate microchromosomes (four pairs) and the microchromosomes (29 pairs). Whilst the microchromosome group comprise only 25% of the DNA, increasing evidence is proving that this is disproportionate to their gene content. This paper demonstrates the utility of cosmid sequence scanning as a potential method for analysing the chicken genome, providing an economical method for the production of a molecular map. The GC content, gene density and repeat distribution are analysed relative to chromosomal origin. Results indicate that gene density is higher on the microchromosomes. During the scanning process an example of conserved linkage between chicken and human (12q34.2) has been demonstrated.

The identification and characterization of microsatellites in the compact genome of the Japanese pufferfish, Fugu rubripes: perspectives in functional and comparative genomic analyses.

Fugu rubripes (Fugu) has one of the smallest recorded vertebrate genomes and is an economic tool for comparative DNA sequence analysis. Initial characterization of 128 kb of Fugu DNA attributed the compactness of this genome, in part, to a sparseness of repetitive DNA sequence compared with mammalian genomic sequences. This paper describes a new and comprehensive analysis in which 501 theoretically possible microsatellites with a repeat unit of one to six bases were used to query two orders of magnitude more Fugu DNA (i.e. 11.338 Mb). A total of 6042 microsatellites were identified and categorized. In decreasing order, the 20 most frequently occurring microsatellites are AC, A, C, AGG, AG, AGC, AAT, AAAT, ACAG, ACGC, ATCC, AAC, ATC, AGGG, AAAG, AAG, AAAC, AT, CCG and TTAGGG. The 20 most frequently occurring microsatellites represent 81.79% of all microsatellites identified. Our results indicate that one microsatellite occurs every 1.876 kb of DNA in Fugu, 11.55% of the microsatellites are detected in open reading frames that are predicted protein coding regions. With respect to the proportion of microsatellites present in open reading frames and the total abundance (bp) of all microsatellites, the genome of Fugu is similar to the genome of many other vertebrate species. Previous estimates performed indicate that approximately 1% of many vertebrate genomes are comprized of microsatellite sequences. However, many differences prevail in the abundance and frequency of the individual microsatellite classes. Many of the frequently occurring microsatellites in Fugu are known to code in other species for regions in proteins such as transcription factors, whilst others are associated with known functions, such as transcription factor binding sites and form part of promoter regions in DNA sequences of genes. Therefore, it is likely that such repeats in genomes have a role in the evolution of genes, regulation of gene expression and consequently the evolution of species.

Assessment of protein fold predictions from sequence information: the predicted alpha/beta doubly wound fold of the von Willebrand factor type A domain is similar to its crystal structure.

The fold of the von Willebrand Factor type A domain (vWF-A) was predicted to be similar to an alpha/beta doubly wound fold in the GTP-binding domain of ras-p21, despite the lack of sequence or functional similarity. This was subsequently confirmed by the vWF-A crystal structure from complement receptor type 3. The prediction is now reviewed. The vWF-A secondary structure was predicted with 62 to 75% accuracy and 12 of the 13 secondary structure elements were identified correctly. Accessibility predictions were 69 to 71% accurate. The fold recognition analysis was confirmed, but was much improved by averaging the results from 70 complete vWF-A sequences. The related folds of ras-P21 and flavodoxin scored highly. In addition, both the mapping of the predicted vWF-A secondary structure elements with those in 12 known alpha/beta folds and two Asp residues at the C-terminal ends of two adjacent beta-strands matched well with ras-p21 and flavodoxin. The predicted Mg(2+)-binding site, two disulphide bridges and the secondary structure topology were largely accurate. The exception is the reversal of a beta-hairpin at one end of the central beta-sheet. We conclude that non-homologous folds with dissimilar functions can be predicted from sequence data with reasonable accuracy, and that the accuracy in this case was principally limited at the periphery of the fold.

The protein fold of the von Willebrand factor type A domain is predicted to be similar to the open twisted beta-sheet flanked by alpha-helices found in human ras-p21.

The von Willebrand Factor type A domain is the prototype for a protein superfamily. It possesses no significant sequence similarity to any known protein structure. Secondary structure predictions indicate a largely alternating pattern of six alpha-helices and six beta-strands. A protein fold for this domain is proposed to correspond to a doubly-wound open twisted beta-sheet structure flanked by alpha-helices. Close agreement was found with the GTP-binding domain of human ras-p21, provided that an extra alpha-helix was inserted. The structure of the predicted fold showed high compatibility with the proximate location of two Mg(2+)-binding Asp residues, two disulphide-bridged Cys residues, and other known functional attributes of this domain.