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We have characterized a novel, captured and fully functional viral interleukin (IL)-10 homologue ((OvHV)IL-10) from the gammaherpesvirus ovine herpesvirus 2. Unlike IL-10 homologues from other gammaherpesviruses, the (OvHV)IL-10 peptide sequence was highly divergent from that of the host species. The (OvHV)IL-10 gene is unique amongst virus captured genes in that it has precisely retained the original cellular exon structure, having five exons of similar sizes to the cellular counterparts. However, the sizes of the introns are dramatically reduced. The (OvHV)IL-10 protein was shown to be a non-glycosylated, secreted protein of M(r) 21 000 with a signal peptidase cleavage site between amino acids 26 and 27 of the nascent peptide. Functional assays showed that (OvHV)IL-10, in a similar way to ovine IL-10, stimulated mast cell proliferation and inhibited macrophage inflammatory chemokine production. This is the first example of a captured herpesvirus gene retaining the full cellular gene structure.

Most human cytomegalovirus (HCMV) genes are highly conserved in sequence among strains, but some exhibit a substantial degree of variation. Two of these genes are UL146, which encodes a CXC chemokine, and UL139, which is predicted to encode a membrane glycoprotein. The sequences of these genes were determined from a collection of 184 HCMV samples obtained from Africa, Australia, Asia, Europe, and North America. UL146 is hypervariable throughout, whereas variation in UL139 is concentrated in a sequence encoding a potentially highly glycosylated region. The UL146 sequences fell into 14 genotypes, as did all previously reported sequences. The UL139 sequences grouped into 8 genotypes, and all previously reported sequences fell into a subset of these. There were minor differences among continents in genotypic frequencies for UL146 and UL139, but no clear geographical separation, and identical nucleotide sequences were represented among communities distant from each other. The frequent detection of multiple genotypes indicated that mixed infections are common. For both genes, the degree of divergence was sufficient to preclude reliable sequence alignments between genotypes in the most variable regions, and the mode of evolution involved in generating the genotypes could not be discerned. Within genotypes, constraint appears to have been the predominant mode, and positive selection was detected marginally at best. No evidence was found for linkage disequilibrium. The emerging scenario is that the HCMV genotypes developed in early human populations (or even earlier), becoming established via founder or bottleneck effects, and have spread, recombined and mixed worldwide in more recent times.

The Caliciviridae family comprises positive-sense RNA viruses of medical and veterinary significance. In humans, caliciviruses are a major cause of acute gastroenteritis, while in animals respiratory illness, conjunctivitis, stomatitis, and hemorrhagic disease are documented. Investigation of virus-host interactions is limited by a lack of culture systems for many viruses in this family. Feline calicivirus (FCV), a member of the Vesivirus genus, provides a tractable model, since it may be propagated in cell culture. Feline junctional adhesion molecule 1 (fJAM-1) was recently identified as a functional receptor for FCV. We have analyzed the structure of this virus-receptor complex by cryo-electron microscopy and three-dimensional image reconstruction, combined with fitting of homology modeled high-resolution coordinates. We show that domain 1 of fJAM-1 binds to the outer face of the P2 domain of the FCV capsid protein VP1, inducing conformational changes in the viral capsid. This study provides the first structural view of a native calicivirus-protein receptor complex and insights into the mechanisms of virus attachment and uncoating.

Genome signatures are data vectors derived from the compositional statistics of DNA. The self-organizing map (SOM) is a neural network method for the conceptualisation of relationships within complex data, such as genome signatures. The various parameters of the SOM training phase are investigated for their effect on the accuracy of the resulting output map. It is concluded that larger SOMs, as well as taking longer to train, are less sensitive in phylogenetic classification of unknown DNA sequences. However, where a classification can be made, a larger SOM is more accurate. Increasing the number of iterations in the training phase of the SOM only slightly increases accuracy, without improving sensitivity. The optimal length of the DNA sequence k-mer from which the genome signature should be derived is 4 or 5, but shorter values are almost as effective. In general, these results indicate that small, rapidly trained SOMs are generally as good as larger, longer trained ones for the analysis of genome signatures. These results may also be more generally applicable to the use of SOMs for other complex data sets, such as microarray data.

Whole genome sequences for three Epstein-Barr virus strains (B95-8, GD1 and AG876) were aligned and compared. In addition to known variable loci (including type-specific alleles for the EBNA2, EBNA3A, EBNA3B and EBNA3C genes, plus the EBNA1 and LMP1 genes), seven large-scale regions of lower-level diversity were identified with strains at each in two major groupings. All three possible patterns of strain associations were represented across the seven loci. Tree-building studies supported the existence of two distinct lineages in each case, and occurrence of recombination between lineages therefore has to be invoked to account for the observed genotypes of virus strains.

Two Epstein-Barr virus (EBV) types are known, EBV1 and EBV2, which possess substantially diverged alleles for latency genes EBNA-2, EBNA-3A, EBNA-3B and EBNA-3C but are thought to be otherwise similar. We report the first complete EBV2 genome sequence, for strain AG876, as 172,764 bp. The sequence was interpreted as containing at least 80 protein coding genes. Comparison with the published EBV1 sequence demonstrated that the two sequences are collinear and, outside the known diverged alleles, generally very close. The EBNA-1 gene was identified as another diverged locus, although its variation is believed not to correlate with EBV type. Patterns of substitution between the two genomes presented a wide spectrum of classes of change. No evidence was seen for involvement of B-cell-specific hypermutation systems in generation of the diverged alleles. Overall, genomic comparisons indicated that the two EBV types should be regarded as belonging to the same virus species.

Many proteins exhibit sequence periodicity, often correlated with a visible structural periodicity. The statistical significance of such periodicity can be assessed by means of a chi-squared-based test, with significance thresholds being calculated from shuffled sequences. Comparison of the complete proteomes of 45 species reveals striking differences in the proportion of periodic proteins and the intensity of the most significant periodicities. Eukaryotes tend to have a higher proportion of periodic proteins than eubacteria, which in turn tend to have more than archaea. The intensity of periodicity in the most periodic proteins is also greatest in eukaryotes. By contrast, the relatively small group of periodic proteins in archaea also tend to be weakly periodic compared to those of eukaryotes and eubacteria. Exceptions to this general rule are found in those prokaryotes with multicellular life-cycle phases, e.g., Methanosarcina sp., or Anabaena sp., which have more periodicities than prokaryotes in general, and in unicellular eukaryotes, which have fewer than multicellular eukaryotes. The distribution of significantly periodic proteins in eukaryotes is over a wide range of period lengths, whereas prokaryotic proteins typically have a more limited set of period lengths. This is further investigated by repeating the analysis on the NRL-3D database of proteins of solved structure. Some short-range periodicities are explicable in terms of basic secondary structure, e.g., alpha helices, while middle-range periodicities are frequently found to consist of known short Pfam domains, e.g., leucine-rich repeats, tetratricopeptides or armadillo domains. However, not all can be explained in this way.

Phylogenetic relationships within the subfamily Gammaherpesvirinae of the family Herpesviridae were investigated for three species in the genus Lymphocryptovirus (or gamma1 group) and nine in the genus Rhadinovirus (or gamma2 group). Alignments of amino acid sequences from up to 28 genes were used to derive trees by maximum-likelihood and Bayesian Monte Carlo Markov chain methods. Two problem areas were identified involving an unresolvable multifurcation for a clade within the gamma2 group, and a high divergence for Murid herpesvirus 4 (MHV4). A robust final tree was obtained, which was valid for genes from across the virus genomes and was rooted by reference to previous analyses of the whole family Herpesviridae. This tree comprised four major lineages: the gamma1 group of primate viruses; a clade of artiodactyl gamma2 viruses; a clade of perissodactyl gamma2 viruses; and a clade of gamma2 viruses with a multifurcation at its base and containing Old World and New World primate viruses, Bovine herpesvirus 4 and MHV4. Developing previous work it was proposed, on the basis of similarities between the gammaherpesvirus tree and the tree of corresponding mammalian hosts, that the first three of these major viral lineages arose in a coevolutionary manner with host lineages, while the fourth had its origin in an ancient interspecies transfer. Transfer of dates from mammalian palaeontology then allowed estimation of dates for nodes in the gammaherpesvirus tree.

The phylogeny of reptilian herpesviruses (HVs) relative to mammalian and avian HVs was investigated by using available gene sequences and by alignment of encoded amino acid sequences and derivation of trees by maximum-likelihood and Bayesian methods. Phylogenetic loci were obtained for green turtle HV (GTHV) primarily on the basis of DNA polymerase (POL) and DNA binding protein sequences, and for lung-eye-trachea disease-associated HV (LETV) primarily from its glycoprotein B sequence; both have nodes on the branch leading to recognized species in the Alphaherpesvirinae subfamily and should be regarded as new members of that subfamily. A similar but less well defined locus was obtained for an iguanid HV based on a partial POL sequence. On the basis of short POL sequences (around 60 amino acid residues), it appeared likely that GTHV and LETV belong to a private clade and that three HVs of gerrhosaurs (plated lizards) are associated with the iguanid HV. Based on phylogenetic branching patterns for mammalian HV lineages that mirror those of host lineages, we estimated a date for the HV tree's root of around 400 million years ago. Estimated dates for branching events in the development of reptilian, avian, and mammalian Alphaherpesvirinae lineages could plausibly be accounted for in part but not completely by ancient coevolution of these virus lines with reptilian lineages and with the development of birds and mammals from reptilian progenitors.

The genetic content of wild-type human cytomegalovirus was investigated by sequencing the 235 645 bp genome of a low passage strain (Merlin). Substantial regions of the genome (genes RL1-UL11, UL105-UL112 and UL120-UL150) were also sequenced in several other strains, including two that had not been passaged in cell culture. Comparative analyses, which employed the published genome sequence of a high passage strain (AD169), indicated that Merlin accurately reflects the wild-type complement of 165 genes, containing no obvious mutations other than a single nucleotide substitution that truncates gene UL128. A sizeable subset of genes exhibits unusually high variation between strains, and comprises many, but not all, of those that encode proteins known or predicted to be secreted or membrane-associated. In contrast to unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in one or both of two groups of genes that may influence cell tropism. One comprises UL128, UL130 and UL131A, which putatively encode secreted proteins, and the other contains RL5A, RL13 and UL9, which are members of the RL11 glycoprotein gene family. The case in support of a lack of protein-coding potential in the region between UL105 and UL111A was also strengthened.

Periodicity was quantified in 4289 Escherichia coli K12 confirmed and putative protein sequences, using a simple chi-square technique previously shown to reveal triplet period periodicity in coding DNA. Periodicities were calculated from period n = 2 to period n = 50 in nine different alphabetic representations of the proteins. By comparison with a randomly generated proteome of the same compositional content, the E. coli proteome does not contain a significant excess of periodic proteins. However, 60 proteins do appear to be significantly periodic in at least one alphabetic representation, after Bonferroni correction, at p < 0.01, and 30 at p < 0.001. These are compared with significantly periodic proteins of solved three-dimensional structure, detected by an identical analysis of the sequences from a protein structure database. It is concluded that there is no evidence for the presence of a proteome-wide quasi-periodicity as predicted by the "duplication and divergence" model of protein evolution and that the major periodicity detected is a consequence of the repetitive tendencies within alpha-helices. However, it is not possible to explain all sequence periodicities in terms of observable secondary structure, as in cases where sequence periodicity can be compared to solved structure, there is often no structural regularity that would provide an obvious explanation in terms of natural selection on protein function.

Copyright 1998 Academic Press Limited

This paper presents use of spreadsheet software to derive various statistics on nucleotide and codon distribution and frequency from gene sequence data, including chi 2 test and their derivatives, and proportion of nucleotides in the third position. The basic principles can be easily extended to other more complex functions. In addition, it can be used to translate a nucleic acid sequence into a protein sequence or to produce a codon usage table. This adaptation permits sequence analysis without expensive dedicated software and easy modification of the analysis according to the user's requirements. It is also ideal for introducing biological science students to the programming potential of spreadsheets.

The genomic GC contents of both nitrogen-fixing and non-fixing members of eight genera of bacteria are investigated. Analysis by t-tests showed that in the two aerobic general investigated (Aquaspirillum and Vibrio) there is a significantly higher GC content in the nitrogen-fixing members of the genus than in those unable to fix nitrogen, whilst in aerobic genera there is either no GC bias, or in the case of two genera (Rhodospirillum and Clostridium) there is a significantly higher GC content in the non-fixing organisms. This suggests that, in many genera, directional mutational pressures are different in nitrogen-fixing and non-fixing organisms. These results are discussed in the light of known mechanisms of mutation pressure and their relation to environmental variables.

Preferential codon usage within synonymous groups (codon bias), is hypothesised to be a consequence of either mutational pressure or translational selection. This paper examines PLP and DM-20, two transcripts differentially spliced from the plp gene, using a variety of previously described indices measuring codon bias as well as a novel index for quantifying the response to directional mutation pressure. The results demonstrate that small regions of extreme codon bias may have appreciable effects on the quantification of total bias, and that this effect may be either an increase or a decrease depending which index is used.

N-acetyl cysteine is an agent which has been shown to interrupt signal transduction processes linking a wide range of stimuli to the activation of NF-kappa B in mammalian cells. We have investigated its effect on the early development of Xenopus embryos by injecting it into blastulae, using concentrations comparable to those effective on cultured cells. High concentrations at the late blastula or early gastrula stage suppress posterior and enhance anterior development, yielding embryos with enlarged cement glands and otherwise consisting of little except head in extreme cases. Reducing the amount of N-acetyl cysteine injected leads to progressively more posterior structures developing. Injection into one- or two-cell embryos gives similar phenotypes, but of reduced severity and the cement gland is not so enlarged. Explants of animal cap cells taken several hours after injection develop to give large amounts of cement gland material. We have examined the expression of a number of genes in the anteriorised embryos. Posterior markers and Xsna are reduced. Noggin and Goosecoid mRNA are up-regulated through the gastrula and persist at these levels until at least the late neurula stage, whereas in controls Noggin is much lower and Goosecoid is absent at these stages. The most anteriorised phenotype may be a consequence of this changed expression.

High level over-expression of XrelA1, a homologue of the p65 sub-unit of NF-kappa B and of Drosophila dorsal, arrests Xenopus development at the gastrula stage, producing a reduction in the levels of expression of various genes of developmental interest without general reduction in transcription or cessation of cell division. There is little Goosecoid expression, even though a dorsal lip forms. At lower levels XrelA1 mRNA primarily produces disruption of the mid-dorsal axis. A dominant interference gene product, delta 222, produces mainly posterior, but also anterior abnormalities. On the basis of these results we postulate that the role of XrelA1 in the vertebrate embryo is unlikely to be in dorsoventral development, but more likely in the formation of the termini.

A considerable quantity of data has been generated using the technique of in vivo gene knockout in mice, much of which is of relevance to the developmental biologist. Null mutations in Hox genes at the 3'-end of the clusters create complex irregularities at the rostral end of the embryo, including defects in the middle ear and the large blood vessels, suggesting that Hox genes may be involved in pattern specification of these structures in addition to the anteroposterior axis. Null mutations in oncogenes either cause wide pleiotropic effects, or act in a restricted manner on the haematopoietic system. Null mutations in growth factors and related molecules cause failure of proliferation in restricted areas of the embryo in some cases, but have little phenotype in others. There is as yet no null mutation which supports the idea that growth factors are involved in mesoderm induction in mammals. A surprising variety of genes have no null phenotype, or one less severe than might have been previously predicted on the basis of their known function in vitro and pattern of expression. This leads to the possibility that genetic redundancy exists in development.

This paper reports the first description of somitogenesis in a non-aquatic-developing amphibian, the Andean marsupial frog, Gastrotheca riobambae. This frog develops from an embryonic disk located on top of a large yolky egg, bearing some resemblance to the embryo of the chick. Besides the histological characterization of somite formation, we quantified cell number in the developing somites of Gastrotheca, Xenopus laevis and the chick. Gastrotheca was found to have a mode of somitogenesis which has previously been encountered in the aquatic-developing toad Bombina. Somitic cell number was found to be an order of magnitude higher than that of Xenopus, and approximately double that of the chick. We discuss the possible relation between mode of somitogenesis, somitic cell number, speed of development and egg size.

To date, three mammalian TGF-beta isoforms have been identified, each encoded by different genetic loci. Through each is very similar in primary amino acid structure, there are clear differences both in the mature bioactive peptide region and in the latency-associated peptide, which could potentially confer differential biological specificity. As one route to investigate differential biological function in vivo we have used gene specific probes for in situ hybridization studies to examine the distribution of RNA transcripts during mammalian embryogenesis. Mouse embryos from 6 to 14.5 gestational age and human embryos from 32 to 57 days post-fertilization have been probed. A general conclusion from these studies is that each TGF-beta gene has a distinct, through overlapping, pattern of transcript distribution and that this pattern, in most cases, is conserved between mouse and man. We have focused on the biological function the TGF-betas play in certain epithelia and in cardiogenesis, which will be discussed in this presentation.

Type beta transforming growth factors (TGF beta s) are members of a large superfamily of related proteins, each of which plays a pivotal role in embryonic processes. The TGF beta s per se are at least five in number, though only three isoforms have been identified in mammals. Here we will review the evidence, taken from in vitro studies on bioactivity and histochemical localization of RNAs and encoded proteins in vivo, that TGF beta 1, beta 2 and beta 3 are involved in several mammalian developmental processes, including control of growth, differentiation, tissue inductions and morphogenesis.